Signal transduction stress-related proteins and methods of use in plants

ABSTRACT

A transgenic plant transformed by a Signal Transduction Stress-Related Protein (STSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated STSRPs, and isolated nucleic acid coding STSRPs, and vectors and host cells containing the latter.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No.09/828,447, filed Apr. 6, 2001 and now U.S. Pat. No. 6,720,477, and thisapplication is copending with U.S. patent application Ser. No.11/566,246, filed Dec. 4, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/770,225, filed Feb. 2, 2004 and now U.S. Pat.No. 7,166,767, which is a divisional of U.S. Nonprovisional patentapplication Ser. No. 09/828,447, which claims the priority benefit ofU.S. Provisional Application Ser. No. 60/196,001 filed Apr. 7, 2000.Each application identified above is hereby incorporated herein in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nucleic acid sequences encodingproteins that are associated with abiotic stress responses and abioticstress tolerance in plants. In particular, this invention relates tonucleic acid sequences encoding proteins that confer drought, cold,and/or salt tolerance to plants.

2. Background Art

Abiotic environmental stresses, such as drought stress, salinity stress,heat stress, and cold stress, are major limiting factors of plant growthand productivity. Crop losses and crop yield losses of major crops suchas rice, maize (corn) and wheat caused by these stresses represent asignificant economic and political factor and contribute to foodshortages in many underdeveloped countries.

Plants are typically exposed during their life cycle to conditions ofreduced environmental water content. Most plants have evolved strategiesto protect themselves against these conditions of desiccation. However,if the severity and duration of the drought conditions are too great,the effects on plant development, growth and yield of most crop plantsare profound. Furthermore, most of the crop plants are very susceptibleto higher salt concentrations in the soil. Continuous exposure todrought and high salt causes major alterations in the plant metabolism.These great changes in metabolism ultimately lead to cell death andconsequently yield losses.

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold and salttolerance in model, drought- and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

Therefore, what is needed is the identification of the genes andproteins involved in these multi-component processes leading to stresstolerance. Elucidating the function of genes expressed in stresstolerant plants will not only advance our understanding of plantadaptation and tolerance to environmental stresses, but also may provideimportant information for designing new strategies for crop improvement.

Expression and function of abiotic stress-inducible genes have been wellstudied at a molecular level. Complex mechanisms seem to be involved ingene expression and signal transduction in response to the stress. Theseinclude the sensing mechanisms of abiotic stress, modulation of thestress signals to cellular signals, transduction to the nucleus, secondmessengers involved in the stress signal transduction, transcriptionalcontrol of stress-inducible genes and the function and cooperation ofstress-inducible genes.

In animal cells, phosphatidylinositol-specific phospholipase C (PI-PLC)plays a key role in early stages of various signal-transductionpathways. Extracellular stimuli such as hormones and growth factorsactivate PI-PLCs. PI-PLC hydrolyzes phosphatidylinositol 4,5-biphosptate(PIP₂) and generates two second messengers, inosito 1,4,5-triphosphtate(IP₃) and 1,2-diacylglycerol (DG). IP₃ induces the release ofintracellular Ca²⁺ into the cytoplasm, which in turn causes variousresponses therein. DG and PIP₂ also function as second messengers andcontrol various cellular responses.

In plants, similar systems are thought to function in abiotic stressresponse. It is clearly demonstrated that phospholipases A, C or D (PLA,PLC or PLD), depending upon their site of cleavage, play a role in theearly signal transduction events that promote the cell volume changesassociated with osmotic stress and osmoregulation in plants which isimportant for plant stress tolerance (Wang X. et at., 2000, BiochemicalSociety Transactions. 28; 813-816; Chapman K D, 1998 Tre. Plant Sci.3:419-426). For example, in guard cells, abscisic acid (ABA)-inducedstomatal closure is mediated by rapid activation of PIP2-PLC. This leadsto an increase in IP₃ levels, a rise in cytosolic calcium, and thesubsequent inhibition of K+ channels. Recently, a gene for phospholipaseC, AtPLC was demonstrated to be rapidly induced by drought and saltstresses in Arabidopsis thaliana (Hirayama, T. et al., 1995 Proc. Natl.Acad. Sci. 92:3903-3907).

As mentioned above, Ca²⁺ ions play important roles as second messengersin various signal-transduction pathways in plants. Marked increase inintracellular Ca²⁺ concentration has been observed upon stimulation bywind, touch, abiotic stresses (cold, drought and salinity) or fungalelicitors. Several genes for Ca²⁺ binding proteins with a conservedEF-hand domain have been isolated and showed increased expression levelupon abiotic stress treatment (Frandsen G. et al., 1996 J Biol. Chem.271:343-348; Takahashi S. et al., 2000 Pant Cell Physiol. 41:898-903).

The enigmatically named 14-3-3 proteins have been also the subject ofconsiderable attention in recent years since they have been implicatedin the regulation of diverse physiological processes in eukaryotesranging from slime moulds to higher plants. In plants, many biologicalroles for 14-3-3 proteins have been suggested. The most significant ofthese include roles in the import of nuclear encoded chloroplastproteins, in the assembly of transcription factor complexes and in theregulation of enzyme activity in response to intracellular signaltransduction cascades (Chung H J. et al., 1999 Tre. Plant Sci.4:367-371). The native 14-3-3 proteins are homo- or heterodimers and, aseach monomer has a binding site, a dimer can potentially bind twotargets, promoting their association. Alternatively, target proteins mayhave more than one 14-3-3-binding site.

Several functions have been proposed for the 14-3-3 proteins in terms ofinvolvement of plant stress tolerance. The 14-3-3 proteins couldfunction as regulators in stress signal transduction. For example,RCI14A and RCI14B genes are induced by cold treatment in Arabidopsis andare highly homologous to the 14-3-3 proteins. The rise in the RCItranscript levels observed in response to cold treatment suggests a rolefor the RCI proteins in the stress signaling transduction pathway(Jarillo J A et al., 1994 Plant Mol. Biol. 25:693-704)

Due to the commercial consequences of environmental damage to crops,there is an interest in understanding the stress response signaltransduction mechanisms in plants and how these can be manipulated toimprove a plant's response to environmental damage. There is a need,therefore, to identify genes expressed in stress tolerant plants thathave the capacity to confer stress resistance to its host plant and toother plant species. Newly generated stress tolerant plants will havemany advantages, such as increasing the range that crop plants can becultivated by, for example, decreasing the water requirements of a plantspecies.

SUMMARY OF THE INVENTION

This invention fulfills in part the need to identify new, unique signaltransduction proteins capable of conferring stress tolerance to plantsupon over-expression. The present invention provides a transgenic plantcell transformed by a Signal Transduction Stress-Related Protein (STSRP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe plant cell results in increased tolerance to environmental stress ascompared to a wild type variety of the plant cell. Namely, describedherein are the transcription factors 1) Phospholipase C-1 (PLC-1); 2)Phospholipase C-2 (PLC-2); 3) 14-3-3 Protein-1 (14-3-3P-1); 4) 14-3-3Protein-1 (14-3-3P-2); and 5) Ca²⁺ Binding Protein-1 (CBP-1), all fromPhyscomitrella patens.

The invention provides in some embodiments that the STSRP and codingnucleic acid are that found in members of the genus Physcomitrella. Inanother preferred embodiment, the nucleic acid and protein are from aPhyscomitrella patens. The invention provides that the environmentalstress can be salinity, drought, temperature, metal, chemical,pathogenic and oxidative stresses, or combinations thereof. In preferredembodiments, the environmental stress can be drought or coldtemperature.

The invention further provides a seed produced by a transgenic planttransformed by a STSRP coding nucleic acid, wherein the plant is truebreeding for increased tolerance to environmental stress as compared toa wild type variety of the plant. The invention further provides a seedproduced by a transgenic plant expressing a STSRP, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant.

The invention further provides an agricultural product produced by anyof the below-described transgenic plants, plant parts or seeds. Theinvention further provides an isolated STSRP as described below. Theinvention further provides an isolated STSRP coding nucleic acid,wherein the STSRP coding nucleic acid codes for a STSRP as describedbelow.

The invention further provides an isolated recombinant expression vectorcomprising a STSRP coding nucleic acid as described below, whereinexpression of the vector in a host cell results in increased toleranceto environmental stress as compared to a wild type variety of the hostcell. The invention further provides a host cell containing the vectorand a plant containing the host cell.

The invention further provides a method of producing a transgenic plantwith a STSRP coding nucleic acid, wherein expression of the nucleic acidin the plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant comprising: (a)transforming a plant cell with an expression vector comprising a STSRPcoding nucleic acid, and (b) generating from the plant cell a transgenicplant with an increased tolerance to environmental stress as compared toa wild type variety of the plant. In preferred embodiments, the STSRPand STSRP coding nucleic acid are as described below.

The present invention further provides a method of identifying a novelSTSRP, comprising (a) raising a specific antibody response to a STSRP,or fragment thereof, as described above; (b) screening putative STSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel STSRP; and(c) identifying from the bound material a novel STSRP in comparison toknown STSRP. Alternatively, hybridization with nucleic acid probes asdescribed below can be used to identify novel STSRP nucleic acids.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a STSRP inthe plant, wherein the STSRP is as described below. The inventionprovides that this method can be performed such that the stresstolerance is either increased or decreased. Preferably, stress toleranceis increased in a plant via increasing expression of a STSRP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the plant expression vector pBPSSC022containing the super promoter driving the expression of SEQ ID NO: 6, 7,8, 9, or 10 (“Desired Gene”). The components are: NPTII kanamycinresistance gene (Bevan M, Nucleic Acids Res. 26: 8711-21, 1984),AtAct2-i promoter (An Y Q et al., Plant J 10: 107-121 1996), OCS3terminator (During K, Transgenic Res. 3: 138-140, 1994), NOSpAterminator (Jefferson et al., EMBO J 6:3901-7 1987).

FIG. 2 shows the results of a drought stress test with over-expressingPpPLC-1 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 3 shows the results of a drought stress test with over-expressingPpPLC-2 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 4 shows the results of a drought stress test with over-expressingPp14-3-3P-1 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 5 shows the results of a drought stress test with over-expressingPp14-3-3P-2 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 6 shows the results of a drought stress test with over-expressingPpCBP-1 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 7 shows the results of a freezing stress test with over-expressingPpPLC-2 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 8 shows the results of a freezing stress test with over-expressingPp14-3-3P-1 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

FIG. 9 shows the results of a freezing stress test with over-expressingPpCBP-1 transgenic plants and wild-type Arabidopsis lines. Thetransgenic lines display a tolerant phenotype. Individual transformantlines are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood thatthe terminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. In particular, thedesignation of the amino acid sequences as protein “Signal TransductionStress-Related Proteins” (STSRPs), in no way limits the functionality ofthose sequences.

The present invention provides a transgenic plant cell transformed by aSTSRP coding nucleic acid, wherein expression of the nucleic acidsequence in the plant cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcell. The invention further provides transgenic plant parts andtransgenic plants containing the plant cells described herein. Alsoprovided is a plant seed produced by a transgenic plant transformed by aSTSRP coding nucleic acid, wherein the seed contains the STSRP codingnucleic acid, and wherein the plant is true breeding for increasedtolerance to environmental stress as compared to a wild type variety ofthe plant. The invention further provides a seed produced by atransgenic plant expressing a STSRP, wherein the seed contains theSTSRP, and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention also provides an agricultural product produced by any ofthe below-described transgenic plants, plant parts and plant seeds.

As used herein, the term “variety” refers to a group of plants within aspecies that share constant characters that separate them from thetypical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more DNA sequences introduced into aplant variety.

The present invention describes for the first time that thePhyscomitrella patens STSRPs, PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 andCBP-1, are useful for increasing a plant's tolerance to environmentalstress. Accordingly, the present invention provides isolated STSRPsselected from the group consisting of PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2and CBP-1, and homologs thereof. In preferred embodiments, the STSRP isselected from 1) a Phospholipase C-1 (PLC-1) protein as defined in SEQID NO:11; 2) a Phospholipase C-2 (PLC-2) protein as defined in SEQ IDNO:12; 3) a 14-3-3 Protein-1 (14-3-3P-1) protein as defined in SEQ IDNO:13; 4) a 14-3-3 Protein-1 (14-3-3P-2) protein as defined in SEQ IDNO: 14; and 5) a Ca²⁺ Binding Protein-1 (CBP-1) protein as defined inSEQ ID NO:15, and homologs and orthologs thereof. Homologs and orthologsof the amino acid sequences are defined below.

The STSRPs of the present invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedabove), the expression vector is introduced into a host cell (asdescribed above) and the STSRP is expressed in the host cell. The STSRPcan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a STSRP polypeptide, or peptide can besynthesized chemically using standard peptide synthesis techniques.Moreover, native STSRP can be isolated from cells (e.g., Physcomitrellapatens), for example using an anti-STSRP antibody, which can be producedby standard techniques utilizing a STSRP or fragment thereof.

The invention further provides an isolated STSRP coding nucleic acid.The present invention includes STSRP coding nucleic acids that encodeSTSRPs as described herein. In preferred embodiments, the STSRP codingnucleic acid is selected from 1) a Phospholipase C-1 (PLC-1) nucleicacid as defined in SEQ ID NO:6; 2) a Phospholipase C-2 (PLC-2) nucleicacid as defined in SEQ ID NO:7; 3) a 14-3-3 Protein-1 (14-3-3P-1)nucleic acid as defined in SEQ ID NO:8; 4) a 14-3-3 Protein-1(14-3-3P-2) nucleic acid as defined in SEQ ID NO:9; and 5) a Ca²⁺Binding Protein-1 (CBP-1) nucleic acid as defined in SEQ ID NO:10 andhomologs and orthologs thereof. Homologs and orthologs of the nucleotidesequences are defined below. In one preferred embodiment, the nucleicacid and protein are isolated from the plant genus Physcomitrella. Inanother preferred embodiment, the nucleic acid and protein are from aPhyscomitrella patens (P. patens) plant.

As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, or temperature, or combinations thereof, and inparticular, can be high salinity, low water content or low temperature.It is also to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

As also used herein, the terms “nucleic acid” and “nucleic acidmolecule” are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of some of the sequences which naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated STSRP nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived (e.g., a Physcomitrella patens cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can be freefrom some of the other cellular material with which it is naturallyassociated, or culture medium when produced by recombinant techniques,or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, or a portion thereof, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. For example, a P. patens STSRP cDNA can beisolated from a P. patens library using all or portion of one of thesequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQID NO:5. Moreover, a nucleic acid molecule encompassing all or a portionof one of the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:5 can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this sequence. Forexample, mRNA can be isolated from plant cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the nucleotide sequences shown in SEQ IDNO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. A nucleicacid molecule of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a STSRP nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. ThesecDNAs comprise sequences encoding the STSRPs (i.e., the “coding region”,indicated in Table 1), as well as 5′ untranslated sequences and 3′untranslated sequences. It is to be understood that SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 comprise both codingregions and 5′ and 3′ untranslated regions. Alternatively, the nucleicacid molecules of the present invention can comprise only the codingregion of any of the sequences in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9 or SEQ ID NO:10 or can contain whole genomic fragmentsisolated from genomic DNA. A coding region of these sequences isindicated as “ORF position”. The present invention also includes STSRPcoding nucleic acids that encode STSRPs as described herein. Preferredis a STSRP coding nucleic acid that encodes a STSRP selected from thegroup consisting of, PLC-1 (SEQ ID NO:11); PLC-2 (SEQ ID NO:12);14-3-3P-1 (SEQ ID NO:13); 14-3-3P-2 (SEQ ID NO:14) and CBP-1 (SEQ IDNO:15).

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, for example, afragment which can be used as a probe or primer or a fragment encoding abiologically active portion of a STSRP. The nucleotide sequencesdetermined from the cloning of the STSRP genes from P. patens allow forthe generation of probes and primers designed for use in identifyingand/or cloning STSRP homologs in other cell types and organisms, as wellas STSRP homologs from other mosses and related species.

Portions of proteins encoded by the STSRP nucleic acid molecules of theinvention are preferably biologically active portions of one of theSTSRPs described herein. As used herein, the term “biologically activeportion of” a STSRP is intended to include a portion, e.g., adomain/motif, of a STSRP that participates in a stress toleranceresponse in a plant, has an activity as set forth in Table 1, orparticipates in the transcription of a protein involved in a stresstolerance response in a plant. To determine whether a STSRP, or abiologically active portion thereof, can participate in transcription ofa protein involved in a stress tolerance response in a plant, or whetherrepression of a STSRP results in increased stress tolerance in a plant,a stress analysis of a plant comprising the STSRP may be performed. Suchanalysis methods are well known to those skilled in the art, as detailedin Example 7. More specifically, nucleic acid fragments encodingbiologically active portions of a STSRP can be prepared by isolating aportion of one of the sequences in SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14 and SEQ ID NO:15, expressing the encoded portion ofthe STSRP or peptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the STSRP or peptide.

Biologically active portions of a STSRP are encompassed by the presentinvention and include peptides comprising amino acid sequences derivedfrom the amino acid sequence of a STSRP, e.g., an amino acid sequence ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15,or the amino acid sequence of a protein homologous to a STSRP, whichinclude fewer amino acids than a full length STSRP or the full lengthprotein which is homologous to a STSRP, and exhibit at least oneactivity of a STSRP. Typically, biologically active portions (e.g.,peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39,40, 50, 100 or more amino acids in length) comprise a domain or motifwith at least one activity of a STSRP. Moreover, other biologicallyactive portions in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe activities described herein. Preferably, the biologically activeportions of a STSRP include one or more selected domains/motifs orportions thereof having biological activity.

The invention also provides STSRP chimeric or fusion proteins. As usedherein, a STSRP “chimeric protein” or “fusion protein” comprises a STSRPpolypeptide operatively linked to a non-STSRP polypeptide. A STSRPpolypeptide refers to a polypeptide having an amino acid sequencecorresponding to a STSRP, whereas a non-STSRP polypeptide refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the STSRP, e.g., a protein thatis different from the STSRP and is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the STSRP polypeptide and the non-STSRPpolypeptide are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The non-STSRPpolypeptide can be fused to the N-terminus or C-terminus of the STSRPpolypeptide. For example, in one embodiment, the fusion protein is aGST-STSRP fusion protein in which the STSRP sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant STSRPs. In another embodiment, the fusionprotein is a STSRP containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a STSRP can be increased through use of aheterologous signal sequence.

Preferably, a STSRP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A STSRPencoding nucleic acid can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the STSRP.

In addition to fragments and fusion proteins of the STSRPs describedherein, the present invention includes homologs and analogs of naturallyoccurring STSRPs and STSRP encoding nucleic acids in a plant. “Homologs”are defined herein as two nucleic acids or proteins that have similar,or “homologous”, nucleotide or amino acid sequences, respectively.Homologs include allelic variants, orthologs, paralogs, agonists andantagonists of STSRPs as defined hereafter. The term “homolog” furtherencompasses nucleic acid molecules that differ from one of thenucleotide sequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10 (and portions thereof) due to degeneracy of thegenetic code and thus encode the same STSRP as that encoded by thenucleotide sequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9 or SEQ ID NO:10. As used herein a “naturally occurring” STSRPrefers to a STSRP amino acid sequence that occurs in nature. Preferably,a naturally occurring STSRP comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14 and SEQ ID NO:15.

An agonist of the STSRP can retain substantially the same, or a subset,of the biological activities of the STSRP. An antagonist of the STSRPcan inhibit one or more of the activities of the naturally occurringform of the STSRP. For example, the STSRP antagonist can competitivelybind to a downstream or upstream member of the cell membrane componentmetabolic cascade that includes the STSRP, or bind to a STSRP thatmediates transport of compounds across such membranes, therebypreventing translocation from taking place.

Nucleic acid molecules corresponding to natural allelic variants andanalogs, orthologs and paralogs of a STSRP cDNA can be isolated based ontheir identity to the Physcomitrella patens STSRP nucleic acidsdescribed herein using STSRP cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. In an alternative embodiment,homologs of the STSRP can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of the STSRP for STSRPagonist or antagonist activity. In one embodiment, a variegated libraryof STSRP variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of STSRP variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential STSRP sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofSTSRP sequences therein. There are a variety of methods that can be usedto produce libraries of potential STSRP homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene is then ligated into an appropriate expression vector.Use of a degenerate set of genes allows for the provision, in onemixture, of all of the sequences encoding the desired set of potentialSTSRP sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A., 1983 Tetrahedron 39:3;Itakura et al., 1984 Annu. Rev. Biochem. 53:323; Itakura et al., 1984Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the STSRP coding regions can beused to generate a variegated population of STSRP fragments forscreening and subsequent selection of homologs of a STSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a STSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA, which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the STSRP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of STSRP homologs. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify STSRP homologs (Arkin and Yourvan, 1992 PNAS 89:7811-7815;Delgrave et al., 1993 Protein Engineering 6(3):327-331). In anotherembodiment, cell based assays can be exploited to analyze a variegatedSTSRP library, using methods well known in the art. The presentinvention further provides a method of identifying a novel STSRP,comprising (a) raising a specific antibody response to a STSRP, or afragment thereof, as described above; (b) screening putative STSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel STSRP; and(c) analyzing the bound material in comparison to known STSRP, todetermine its novelty.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 and SEQ ID NO:15 and a mutant form thereof), the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe sequence of one protein or nucleic acid for optimal alignment withthe other protein or nucleic acid). The amino acid residues atcorresponding amino acid positions are then compared. When a position inone sequence (e.g., one of the sequences of SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15) is occupied by the sameamino acid residue as the corresponding position in the other sequence(e.g., a mutant form of the sequence selected from the polypeptide ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ IDNO:15), then the molecules are homologous at that position (i.e., asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The same type of comparison can bemade between two nucleic acid sequences.

The percent homology between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %homology=numbers of identical positions/total numbers of positions×100).Preferably, the amino acid sequences included in the present inventionare at least about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-80%, 80-90%, 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 or SEQ ID NO:15. In yet another embodiment, at least about 50-60%,preferably at least about 60-70%, and more preferably at least about70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%,98%, 99% or more homologous to an entire amino acid sequence encoded bya nucleic acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9 or SEQ ID NO:10. In other embodiments, the preferable lengthof sequence comparison for proteins is at least 15 amino acid residues,more preferably at least 25 amino acid residues, and most preferably atleast 35 amino acid residues.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown inSEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or aportion thereof. The preferable length of sequence comparison fornucleic acids is at least 75 nucleotides, more preferably at least 100nucleotides and most preferably the entire length of the coding region.

It is also preferable that the homologous nucleic acid molecule of theinvention encodes a protein or portion thereof which includes an aminoacid sequence which is sufficiently homologous to an amino acid sequenceof SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ IDNO:15 such that the protein or portion thereof maintains the same or asimilar function as the amino acid sequence to which it is compared.Functions of the STSRP amino acid sequences of the present inventioninclude the ability to participate in a stress tolerance response in aplant, or more particularly, to participate in the transcription of aprotein involved in a stress tolerance response in a Physcomitrellapatens plant. Examples of such activities are described in Table 1.

In addition to the above described methods, a determination of thepercent homology between two sequences can be accomplished using amathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA90:5873-5877). Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, et al. (1990 J. Mol. Biol. 215:403-410).

BLAST nucleic acid searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleic acid sequences homologous tothe STSRP nucleic acid molecules of the invention. Additionally, BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to STSRPs of thepresent invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller (CABIOS 1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) that is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 can be used toobtain amino acid sequences homologous to the STSRPs of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997 NucleicAcids Res. 25:3389-3402). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller (CABIOS 1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) that is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 can be used.

Finally, homology between nucleic acid sequences can also be determinedusing hybridization techniques known to those of skill in the art.Accordingly, an isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes, e.g., under stringentconditions, to one of the nucleotide sequences shown in SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, or a portionthereof. More particularly, an isolated nucleic acid molecule of theinvention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising anucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9 or SEQ ID NO:10. In other embodiments, the nucleic acid is at least30, 50, 100, 250 or more nucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, 6.3.1-6.3.6, John Wiley& Sons, N.Y. (1989). A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQID NO:10 corresponds to a naturally occurring nucleic acid molecule. Asused herein, a “naturally occurring” nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein). In one embodiment, the nucleic acidencodes a naturally occurring Physcomitrella patens STSRP.

Using the above-described methods, and others known to those of skill inthe art, one of ordinary skill in the art can isolate homologs of theSTSRPs comprising amino acid sequences shown in SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. One subset of thesehomologs is allelic variants. As used herein, the term “allelic variant”refers to a nucleotide sequence containing polymorphisms that lead tochanges in the amino acid sequences of a STSRP and that exist within anatural population (e.g., a plant species or variety). Such naturalallelic variations can typically result in 1-5% variance in a STSRPnucleic acid. Allelic variants can be identified by sequencing thenucleic acid sequence of interest in a number of different plants, whichcan be readily carried out by using hybridization probes to identify thesame STSRP genetic locus in those plants. Any and all such nucleic acidvariations and resulting amino acid polymorphisms or variations in aSTSRP that are the result of natural allelic variation and that do notalter the functional activity of a STSRP, are intended to be within thescope of the invention.

Moreover, nucleic acid molecules encoding STSRPs from the same or otherspecies such as STSRP analogs, orthologs and paralogs, are intended tobe within the scope of the present invention. As used herein, the term“analogs” refers to two nucleic acids that have the same or similarfunction, but that have evolved separately in unrelated organisms. Asused herein, the term “orthologs” refers to two nucleic acids fromdifferent species, but that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode proteins having the same orsimilar functions. As also used herein, the term “paralogs” refers totwo nucleic acids that are related by duplication within a genome.Paralogs usually have different functions, but these functions may berelated (Tatusov, R. L. et al. 1997 Science 278(5338):631-637). Analogs,orthologs and paralogs of a naturally occurring STSRP can differ fromthe naturally occurring STSRP by post-translational modifications, byamino acid sequence differences, or by both. Post-translationalmodifications include in vivo and in vitro chemical derivatization ofpolypeptides, e.g., acetylation, carboxylation, phosphorylation, orglycosylation, and such modifications may occur during polypeptidesynthesis or processing or following treatment with isolated modifyingenzymes. In particular, orthologs of the invention will generallyexhibit at least 80-85%, more preferably 90%, and most preferably 95%,96%, 97%, 98% or even 99% identity or homology with all or part of anaturally occurring STSRP amino acid sequence and will exhibit afunction similar to a STSRP. Orthologs of the present invention are alsopreferably capable of participating in the stress response in plants. Inone embodiment, the STSRP orthologs maintain the ability to participatein the metabolism of compounds necessary for the construction ofcellular membranes in Physcomitrella patens, or in the transport ofmolecules across these membranes.

In addition to naturally-occurring variants of a STSRP sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofSEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10,thereby leading to changes in the amino acid sequence of the encodedSTSRP, without altering the functional ability of the STSRP. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in a sequence of SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of one of the STSRPs without altering theactivity of said STSRP, whereas an “essential” amino acid residue isrequired for STSRP activity. Other amino acid residues, however, (e.g.,those that are not conserved or only semi-conserved in the domain havingSTSRP activity) may not be essential for activity and thus are likely tobe amenable to alteration without altering STSRP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding STSRPs that contain changes in amino acid residuesthat are not essential for STSRP activity. Such STSRPs differ in aminoacid sequence from a sequence contained in SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, yet retain at least one ofthe STSRP activities described herein. In one embodiment, the isolatednucleic acid molecule comprises a nucleotide sequence encoding aprotein, wherein the protein comprises an amino acid sequence at leastabout 50% homologous to an amino acid sequence of SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. Preferably, theprotein encoded by the nucleic acid molecule is at least about 50-60%homologous to one of the sequences of SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14 and SEQ ID NO:15, more preferably at least about60-70% homologous to one of the sequences of SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, even more preferably atleast about 70-80%, 80-90%, 90-95% homologous to one of the sequences ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15,and most preferably at least about 96%, 97%, 98%, or 99% homologous toone of the sequences of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 and SEQ ID NO:15. The preferred STSRP homologs of the presentinvention are preferably capable of participating in the a stresstolerance response in a plant, or more particularly, participating inthe transcription of a protein involved in a stress tolerance responsein a Physcomitrella patens plant, or have one or more activities setforth in Table 1.

An isolated nucleic acid molecule encoding a STSRP homologous to aprotein sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 or SEQ ID NO:15 can be created by introducing one or morenucleotide substitutions, additions or deletions into a nucleotidesequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ IDNO:10 such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10 by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a STSRP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a STSRP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a STSRP activity described herein to identify mutants that retainSTSRP activity. Following mutagenesis of one of the sequences of SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined by analyzing the stress tolerance of a plantexpressing the protein as described in Example 7.

In addition to the nucleic acid molecules encoding the STSRPs describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire STSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a STSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues (e.g.,the entire coding region of, comprises nucleotides 1 to . . . ). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding a STSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of oneof the nucleotide sequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10, or a portion thereof. A nucleic acidmolecule that is complementary to one of the nucleotide sequences shownin SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10is one which is sufficiently complementary to one of the nucleotidesequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9and SEQ ID NO:10 such that it can hybridize to one of the nucleotidesequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9and SEQ ID NO:10, thereby forming a stable duplex.

Given the coding strand sequences encoding the STSRPs disclosed herein(e.g., the sequences set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9 and SEQ ID NO:10), antisense nucleic acids of the inventioncan be designed according to the rules of Watson and Crick base pairing.The antisense nucleic acid molecule can be complementary to the entirecoding region of STSRP mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof STSRP mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofSTSRP mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a STSRP tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., 1987 Nucleic Acids. Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes described in Haselhoff andGerlach, 1988 Nature 334:585-591) can be used to catalytically cleaveSTSRP mRNA transcripts to thereby inhibit translation of STSRP mRNA. Aribozyme having specificity for a STSRP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a STSRP cDNA, asdisclosed herein (i.e., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9 or SEQ ID NO:10) or on the basis of a heterologous sequence to beisolated according to methods taught in this invention. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a STSRP-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, STSRP mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W., 1993 Science261:1411-1418.

Alternatively, STSRP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a STSRPnucleotide sequence (e.g., a STSRP promoter and/or enhancer) to formtriple helical structures that prevent transcription of a STSRP gene intarget cells. See generally, Helene, C., 1991 Anticancer Drug Des.6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J., 1992 Bioassays 14(12):807-15.

In addition to the STSRP nucleic acids and proteins described above, thepresent invention encompasses these nucleic acids and proteins attachedto a moiety. These moieties include, but are not limited to, detectionmoieties, hybridization moieties, purification moieties, deliverymoieties, reaction moieties, binding moieties, and the like. One typicalgroup of nucleic acids attached to a moiety include probes and primers.The probes and primers comprise a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably about 40, 50 or 75 consecutive nucleotides ofa sense strand of one of the sequences set forth in SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, an anti-sense sequenceof one of the sequences set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10, or naturally occurring mutantsthereof. Primers based on a nucleotide sequence of SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 can be used in PCRreactions to clone STSRP homologs. Probes based on the STSRP nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or homologous proteins. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g. the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor. Such probes can be used as a part of a genomic markertest kit for identifying cells which express a STSRP, such as bymeasuring a level of a STSRP-encoding nucleic acid, in a sample ofcells, e.g., detecting STSRP mRNA levels or determining whether agenomic STSRP gene has been mutated or deleted.

In particular, a useful method to ascertain the level of transcriptionof the gene (an indicator of the amount of mRNA available fortranslation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York). This information at least partiallydemonstrates the degree of transcription of the transformed gene. Totalcellular RNA can be prepared from cells, tissues or organs by severalmethods, all well-known in the art, such as that described in Bormann,E. R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence orrelative quantity of protein translated from this mRNA, standardtechniques, such as a Western blot, may be employed. These techniquesare well known to one of ordinary skill in the art. (See, for example,Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: NewYork).

The invention further provides an isolated recombinant expression vectorcomprising a STSRP nucleic acid as described above, wherein expressionof the vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., STSRPs, mutant forms of STSRPs, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of STSRPs in prokaryotic or eukaryotic cells. For example,STSRP genes can be expressed in bacterial cells such as C. glutamicum,insect cells (using baculovirus expression vectors), yeast and otherfungal cells (see Romanos, M. A. et al., 1992 Foreign gene expression inyeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J. J. et al.,1991 Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P. J.,1991 Gene transfer systems and vector development for filamentous fungi,in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., p.1-28, Cambridge University Press: Cambridge), algae (Falciatore et al.,1999 Marine Biotechnology 1(3):239-251), ciliates of the types:Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in WO 98/01572 and multicellular plant cells (seeSchmidt, R. and Willmitzer, L., 1988 High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants, Plant Cell Rep. 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung and R.Wu, 128-43, Academic Press: 1993; Potrykus, 1991 Annu. Rev. PlantPhysiol. Plant Molec. Biol. 42:205-225 and references cited therein) ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press:San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of a recombinantprotein; 2) to increase the solubility of a recombinant protein; and 3)to aid in the purification of a recombinant protein by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the STSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant STSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11 d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11 d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992 Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

In another embodiment, the STSRP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., 1987 EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987 Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, include those detailedin: van den Hondel, C.A.M.J.J. & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi, in: AppliedMolecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28,Cambridge University Press: Cambridge.

Alternatively, the STSRPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

In yet another embodiment, a STSRP nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987Nature 329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2^(ed) ., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987 Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988 Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989 EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983 Cell 33:729-740; Queen andBaltimore, 1983 Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989 PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al., 1985 Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss, 1990 Science 249:374-379)and the fetoprotein promoter (Campes and Tilghman, 1989 Genes Dev.3:537-546).

In another embodiment, the STSRPs of the invention may be expressed inunicellular plant cells (such as algae) (see Falciatore et al., 1999Marine Biotechnology 1(3):239-251 and references therein) and plantcells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

A plant expression cassette preferably contains regulatory sequencescapable of driving gene expression in plant cells and operably linked sothat each sequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereofbut also all other terminators functionally active in plants aresuitable.

As plant gene expression is very often not limited on transcriptionallevels, a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) orplant promoters like those from Rubisco small subunit described in U.S.Pat. No. 4,962,028.

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene product in itsappropriate cell compartment (for review see Kermode, 1996 Crit. Rev.Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

Plant gene expression can also be facilitated via an inducible promoter(for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is wanted to occur in a time specific manner. Examplesof such promoters are a salicylic acid inducible promoter (PCTApplication No. WO 95/19443), a tetracycline inducible promoter (Gatz etal., 1992 Plant J. 2:397-404) and an ethanol inducible promoter (PCTApplication No. WO 93/21334).

Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (PCT Application No. WO 96/12814) orthe wound-inducible pinII-promoter (European Patent No. 375091). Forother examples of drought, cold, and salt-inducible promoters, such asthe RD29A promoter, see Yamaguchi-Shinozalei et al. (1993 Mol. Gen.Genet. 236:331-340).

Especially preferred are those promoters that confer gene expression inspecific tissues and organs, such as guard cells and the root haircells. Suitable promoters include the napin-gene promoter from rapeseed(U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumleinet al., 1991 Mol Gen Genet. 225(3):459-67), the oleosin-promoter fromArabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoterfrom Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoterfrom Brassica (PCT Application No. WO 91/13980) or the legumin B4promoter (LeB4; Baeumlein et al., 1992 Plant Journal, 2(2):233-9) aswell as promoters conferring seed specific expression in monocot plantslike maize, barley, wheat, rye, rice, etc. Suitable promoters to noteare the lpt2 or lpt1-gene promoter from barley (PCT Application No. WO95/15389 and PCT Application No. WO 95/23230) or those described in PCTApplication No. WO 99/16890 (promoters from the barley hordein-gene,rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadingene, wheat glutelin gene, maize zein gene, oat glutelin gene, Sorghumkasirin-gene and rye secalin gene).

Also especially suited are promoters that confer plastid-specific geneexpression since plastids are the compartment where lipid biosynthesisoccurs. Suitable promoters are the viral RNA-polymerase promoterdescribed in PCT Application No. WO 95/16783 and PCT Application No. WO97/06250 and the clpP-promoter from Arabidopsis described in PCTApplication No. WO 99/46394.

The invention further provides a recombinant expression vectorcomprising a STSRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a STSRP mRNA. Regulatory sequences operativelylinked to a nucleic acid molecule cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986 and Mol et al.,1990 FEBS Letters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aSTSRP can be expressed in bacterial cells such as C. glutamicum, insectcells, fungal cells or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells), algae, ciliates, plant cells, fungi or othermicroorganisms like C. glutamicum. Other suitable host cells are knownto those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual.2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratorymanuals such as Methods in Molecular Biology, 1995, Vol. 44,Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa,N.J. As biotic and abiotic stress tolerance is a general trait wished tobe inherited into a wide variety of plants like maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola,manihot, pepper, sunflower and tagetes, solanaceous plants like potato,tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants(coffee, cacao, tea), Salix species, trees (oil palm, coconut),perennial grasses and forage crops, these crop plants are also preferredtarget plants for a genetic engineering as one further embodiment of thepresent invention.

In particular, the invention provides a method of producing a transgenicplant with a STSRP coding nucleic acid, wherein expression of thenucleic acid(s) in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a STSRP nucleic acid, and (b) generating from the plant cella transgenic plant with a increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention alsoprovides a method of increasing expression of a gene of interest withina host cell as compared to a wild type variety of the host cell, whereinthe gene of interest is transcribed in response to a STSRP, comprising:(a) transforming the host cell with an expression vector comprising aSTSRP coding nucleic acid, and (b) expressing the STSRP within the hostcell, thereby increasing the expression of the gene transcribed inresponse to the STSRP, as compared to a wild type variety of the hostcell.

For such plant transformation, binary vectors such as pBinAR can be used(Höfgen and Willmitzer, 1990 Plant Science 66:221-230). Construction ofthe binary vectors can be performed by ligation of the cDNA in sense orantisense orientation into the T-DNA. 5-prime to the cDNA a plantpromoter activates transcription of the cDNA. A polyadenylation sequenceis located 3-prime to the cDNA. Tissue-specific expression can beachieved by using a tissue specific promoter. For example, seed-specificexpression can be achieved by cloning the napin or LeB4 or USP promoter5-prime to the cDNA. Also, any other seed specific promoter element canbe used. For constitutive expression within the whole plant, the CaMV35S promoter can be used. The expressed protein can be targeted to acellular compartment using a signal peptide, for example for plastids,mitochondria or endoplasmic reticulum (Kermode, 1996 Crit. Rev. PlantSci. 4 (15):285-423). The signal peptide is cloned 5-prime in frame tothe cDNA to archive subcellular localization of the fusion protein.Additionally, promoters that are responsive to abiotic stresses can beused with, such as the Arabidopsis promoter RD29A, the nucleic acidsequences disclosed herein. One skilled in the art will recognize thatthe promoter used should be operatively linked to the nucleic acid suchthat the promoter causes transcription of the nucleic acid which resultsin the synthesis of an mRNA which encodes a polypeptide. Alternatively,the RNA can be an antisense RNA for use in affecting subsequentexpression of the same or another gene or genes.

Alternate methods of transfection include the direct transfer of DNAinto developing flowers via electroporation or Agrobacterium mediatedgene transfer. Agrobacterium mediated plant transformation can beperformed using for example the GV3101(pMP90) (Koncz and Schell, 1986Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacteriumtumefaciens strain. Transformation can be performed by standardtransformation and regeneration techniques (Deblaere et al., 1994 Nucl.Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A,Plant Molecular Biology Manual, 2^(nd) Ed.—Dordrecht: Kluwer AcademicPubl., 1995.—in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in PlantMolecular Biology and Biotechnology, Boca Raton: CRC Press, 1993.—360S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed viacotyledon or hypocotyl transformation (Moloney et al., 1989 Plant cellReport 8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Useof antibiotica for Agrobacterium and plant selection depends on thebinary vector and the Agrobacterium strain used for transformation.Rapeseed selection is normally performed using kanamycin as selectableplant marker. Agrobacterium mediated gene transfer to flax can beperformed using, for example, a technique described by Mlynarova et al.,1994 Plant Cell Report 13:282-285. Additionally, transformation ofsoybean can be performed using for example a technique described inEuropean Patent No. 0424 047, U.S. Pat. No. 5,322,783, European PatentNo. 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770.Transformation of maize can be achieved by particle bombardment,polyethylene glycol mediated DNA uptake or via the silicon carbide fibertechnique. (See, for example, Freeling and Walbot “The maize handbook”Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific exampleof maize transformation is found in U.S. Pat. No. 5,990,387 and aspecific example of wheat transformation can be found in PCT ApplicationNo. WO 93/07256.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate or in plants thatconfer resistance towards a herbicide such as glyphosate or glufosinate.Nucleic acid molecules encoding a selectable marker can be introducedinto a host cell on the same vector as that encoding a STSRP or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid molecule can be identified by, for example, drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of a STSRP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the STSRP gene. Preferably, the STSRP gene is aPhyscomitrella patens STSRP gene, but it can be a homolog from a relatedplant or even from a mammalian, yeast, or insect source. In a preferredembodiment, the vector is designed such that, upon homologousrecombination, the endogenous STSRP gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as aknock-out vector). Alternatively, the vector can be designed such that,upon homologous recombination, the endogenous STSRP gene is mutated orotherwise altered but still encodes a functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous STSRP). To create a point mutation viahomologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

Whereas in the homologous recombination vector, the altered portion ofthe STSRP gene is flanked at its 5′ and 3′ ends by an additional nucleicacid molecule of the STSRP gene to allow for homologous recombination tooccur between the exogenous STSRP gene carried by the vector and anendogenous STSRP gene, in a microorganism or plant. The additionalflanking STSRP nucleic acid molecule is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several hundreds of base pairs up to kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R.,and Capecchi, M. R., 1987 Cell 51:503 for a description of homologousrecombination vectors or Strepp et al., 1998 PNAS, 95 (8):4368-4373 forcDNA based recombination in Physcomitrella patens). The vector isintroduced into a microorganism or plant cell (e.g., via polyethyleneglycol mediated DNA), and cells in which the introduced STSRP gene hashomologously recombined with the endogenous STSRP gene are selectedusing art-known techniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a STSRP gene on a vectorplacing it under control of the lac operon permits expression of theSTSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a STSRP.Accordingly, the invention further provides methods for producing STSRPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a STSRP has been introduced, or into whichgenome has been introduced a gene encoding a wild-type or altered STSRP)in a suitable medium until STSRP is produced. In another embodiment, themethod further comprises isolating STSRPs from the medium or the hostcell.

Another aspect of the invention pertains to isolated STSRPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is free of some of thecellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof STSRP in which the protein is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a STSRP having less thanabout 30% (by dry weight) of non-STSRP material (also referred to hereinas a “contaminating protein”), more preferably less than about 20% ofnon-STSRP material, still more preferably less than about 10% ofnon-STSRP material, and most preferably less than about 5% non-STSRPmaterial.

When the STSRP or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation. The language “substantially free ofchemical precursors or other chemicals” includes preparations of STSRPin which the protein is separated from chemical precursors or otherchemicals that are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of a STSRP having less than about30% (by dry weight) of chemical precursors or non-STSRP chemicals, morepreferably less than about 20% chemical precursors or non-STSRPchemicals, still more preferably less than about 10% chemical precursorsor non-STSRP chemicals, and most preferably less than about 5% chemicalprecursors or non-STSRP chemicals. In preferred embodiments, isolatedproteins, or biologically active portions thereof, lack contaminatingproteins from the same organism from which the STSRP is derived.Typically, such proteins are produced by recombinant expression of, forexample, a Physcomitrella patens STSRP in plants other thanPhyscomitrella patens or microorganisms such as C. glutamicum, ciliates,algae or fungi.

The nucleic acid molecules, proteins, protein homologs, fusion proteins,primers, vectors, and host cells described herein can be used in one ormore of the following methods: identification of Physcomitrella patensand related organisms; mapping of genomes of organisms related toPhyscomitrella patens; identification and localization of Physcomitrellapatens sequences of interest; evolutionary studies; determination ofSTSRP regions required for function; modulation of a STSRP activity;modulation of the metabolism of one or more cell functions; modulationof the transmembrane transport of one or more compounds; and modulationof stress resistance.

The moss Physcomitrella patens represents one member of the mosses. Itis related to other mosses such as Ceratodon purpureus which is capableof growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of homology on the DNA sequence andpolypeptide level allowing the use of heterologous screening of DNAmolecules with probes evolving from other mosses or organisms, thusenabling the derivation of a consensus sequence suitable forheterologous screening or functional annotation and prediction of genefunctions in third species. The ability to identify such functions cantherefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

The STSRP nucleic acid molecules of the invention have a variety ofuses. Most importantly, the nucleic acid and amino acid sequences of thepresent invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold. Thepresent invention therefore provides a transgenic plant transformed by aSTSRP nucleic acid, wherein expression of the nucleic acid sequence inthe plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant. The transgenic plant canbe a monocot or a dicot. The invention further provides that thetransgenic plant can be selected from maize, wheat, rye, oat, triticale,rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot,pepper, sunflower, tagetes, solanaceous plants, potato, tobacco,eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salixspecies, oil palm, coconut, perennial grass and forage crops, forexample.

In particular, the present invention describes using the expression ofPLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 of Physcomitrella patens toengineer drought-tolerant, salt-tolerant and/or cold-tolerant plants.This strategy has herein been demonstrated for Arabidopsis thaliana,Rapeseed/Canola, soybeans, corn and wheat but its application is notrestricted to these plants. Accordingly, the invention provides atransgenic plant containing a STSRP selected from PLC-1 (SEQ ID NO:11);PLC-2 (SEQ ID NO:12); 14-3-3P-1 (SEQ ID NO:13); 14-3-3P-2 (SEQ ID NO:14)and CBP-1 (SEQ ID NO:15), wherein the environmental stress is drought,increased salt or decreased or increased temperature. In preferredembodiments, the environmental stress is drought or decreasedtemperature.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a STSRP inthe plant. The invention provides that this method can be performed suchthat the stress tolerance is either increased or decreased. Inparticular, the present invention provides methods of producing atransgenic plant having an increased tolerance to environmental stressas compared to a wild type variety of the plant comprising increasingexpression of a STSRP in a plant.

The methods of increasing expression of STSRPs can be used wherein theplant is either transgenic or not transgenic. In cases when the plant istransgenic, the plant can be transformed with a vector containing any ofthe above described STSRP coding nucleic acids, or the plant can betransformed with a promoter that directs expression of native STSRP inthe plant, for example. The invention provides that such a promoter canbe tissue specific. Furthermore, such a promoter can be developmentallyregulated. Alternatively, non-transgenic plants can have native STSRPexpression modified by inducing a native promoter.

The expression of PLC-1 (SEQ ID NO:11); PLC-2 (SEQ ID NO:12); 14-3-3P-1(SEQ ID NO:13); 14-3-3P-2 (SEQ ID NO:14) or CBP-1 (SEQ ID NO:15) intarget plants can be accomplished by, but is not limited to, one of thefollowing examples: (a) constitutive promoter, (b) stress-induciblepromoter, (c) chemical-induced promoter, and (d) engineered promoterover-expression with for example zinc-finger derived transcriptionfactors (Greisman and Pabo, 1997 Science 275:657). The later caseinvolves identification of the PLC-1 (SEQ ID NO:11); PLC-2 (SEQ IDNO:12); 14-3-3P-1 (SEQ ID NO:13); 14-3-3P-2 (SEQ ID NO:14) or CBP-1 (SEQID NO:15) homologs in the target plant as well as from its promoter.Zinc-finger-containing recombinant transcription factors are engineeredto specifically interact with the PLC-1 (SEQ ID NO:11); PLC-2 (SEQ IDNO:12); 14-3-3P-1 (SEQ ID NO:13); 14-3-3P-2 (SEQ ID NO:14) or CBP-1 (SEQID NO:15) homolog and transcription of the corresponding gene isactivated.

In addition to introducing the STSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Physcomitrella patens or a close relative thereof.Also, they may be used to identify the presence of Physcomitrella patensor a relative thereof in a mixed population of microorganisms. Theinvention provides the nucleic acid sequences of a number ofPhyscomitrella patens genes; by probing the extracted genomic DNA of aculture of a unique or mixed population of microorganisms understringent conditions with a probe spanning a region of a Physcomitrellapatens gene which is unique to this organism, one can ascertain whetherthis organism is present.

Further, the nucleic acid and protein molecules of the invention mayserve as markers for specific regions of the genome. This has utilitynot only in the mapping of the genome, but also in functional studies ofPhyscomitrella patens proteins. For example, to identify the region ofthe genome to which a particular Physcomitrella patens DNA-bindingprotein binds, the Physcomitrella patens genome could be digested, andthe fragments incubated with the DNA-binding protein. Those fragmentsthat bind the protein may be additionally probed with the nucleic acidmolecules of the invention, preferably with readily detectable labels.Binding of such a nucleic acid molecule to the genome fragment enablesthe localization of the fragment to the genome map of Physcomitrellapatens, and, when performed multiple times with different enzymes,facilitates a rapid determination of the nucleic acid sequence to whichthe protein binds. Further, the nucleic acid molecules of the inventionmay be sufficiently homologous to the sequences of related species suchthat these nucleic acid molecules may serve as markers for theconstruction of a genomic map in related mosses.

The STSRP nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic and transportprocesses in which the molecules of the invention participate areutilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein that are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

Manipulation of the STSRP nucleic acid molecules of the invention mayresult in the production of STSRPs having functional differences fromthe wild-type STSRPs. These proteins may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a STSRP ofthe invention may directly affect stress response and/or stresstolerance. In the case of plants expressing STSRPs, increased transportcan lead to improved salt and/or solute partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which export ionic molecules from the cell, it maybe possible to affect the salt tolerance of the cell.

The effect of the genetic modification in plants, C. glutamicum, fungi,algae, or ciliates on stress tolerance can be assessed by growing themodified microorganism or plant under less than suitable conditions andthen analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, protein synthesis, carbohydratesynthesis, lipid synthesis, evapotranspiration rates, general plantand/or crop yield, flowering, reproduction, seed setting, root growth,respiration rates, photosynthesis rates, etc. (Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III:Product recovery and purification, page 469-714, VCH: Weinheim; Belter,P. A. et al., 1988 Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J.M.S.,1992 Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in:Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived there from can then be assayedfor fail or alteration of their tolerance to drought, salt, andtemperature stress.

The engineering of one or more STSRP genes of the invention may alsoresult in STSRPs having altered activities which indirectly impact thestress response and/or stress tolerance of algae, plants, ciliates orfungi or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more STSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells (Girke, T., 1998 The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation see U.S. Pat. No.6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999Spliceosome-mediated RNA trans-splicing as a tool for gene therapyNature Biotechnology 17:246-252.

The aforementioned mutagenesis strategies for STSRPs resulting inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and protein molecules of the invention may beutilized to generate algae, ciliates, plants, fungi or othermicroorganisms like C. glutamicum expressing mutated STSRP nucleic acidand protein molecules such that the stress tolerance is improved.

The present invention also provides antibodies that specifically bind toa STSRP, or a portion thereof, as encoded by a nucleic acid describedherein. Antibodies can be made by many well-known methods (See, e.g.Harlow and Lane, “Antibodies; A Laboratory Manual” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigencan be injected into an animal in an amount and in intervals sufficientto elicit an immune response. Antibodies can either be purifieddirectly, or spleen cells can be obtained from the animal. The cells canthen fused with an immortal cell line and screened for antibodysecretion. The antibodies can be used to screen nucleic acid clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced. (See, for example, Kelly et al., 1992 Bio/Technology10:163-167; Bebbington et al., 1992 Bio/Technology 10:169-175).

The phrases “selectively binds” and “specifically binds” with thepolypeptide refer to a binding reaction that is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bound to a particular protein do not bind in asignificant amount to other proteins present in the sample. Selectivebinding of an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats may be used to select antibodies that selectivelybind with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies selectivelyimmunoreactive with a protein. See Harlow and Lane “Antibodies, ALaboratory Manual” Cold Spring Harbor Publications, New York, (1988),for a description of immunoassay formats and conditions that could beused to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., editors, “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane(“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, NewYork, 1988).

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1

Growth of Physcomitrella patens Cultures

For this study, plants of the species Physcomitrella patens (Hedw.)B.S.G. from the collection of the genetic studies section of theUniversity of Hamburg were used. They originate from the strain 16/14collected by H.L.K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55, 438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

Culturing was carried out in a climatic chamber at an air temperature of25° C. and light intensity of 55 micromol s⁻¹m⁻² (white light; PhilipsTL 65W/25 fluorescent tube) and a light/dark change of 16/8 hours. Themoss was either modified in liquid culture using Knop medium accordingto Reski and Abel (1985, Planta 165:354-358) or cultured on Knop solidmedium using 1% oxoid agar (Unipath, Basingstoke, England). Theprotonemas used for RNA and DNA isolation were cultured in aeratedliquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2 Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of plant material. The materials used include thefollowing buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar togive a fine powder and transferred to 2 ml Eppendorf vessels. The frozenplant material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl ofβ-mercaptoethanol and 10 μl of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000×gand room temperature for 15 minutes in each case. The DNA was thenprecipitated at −70° C. for 30 minutes using ice-cold isopropanol. Theprecipitated DNA was sedimented at 4° C. and 10,000 g for 30 minutes andresuspended in 180 μl of TE buffer (Sambrook et al., 1989, Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6). For further purification,the DNA was treated with NaCl (1.2 M final concentration) andprecipitated again at −70° C. for 30 minutes using twice the volume ofabsolute ethanol. After a washing step with 70% ethanol, the DNA wasdried and subsequently taken up in 50 μl of H₂O+RNAse (50 mg/ml finalconcentration). The DNA was dissolved overnight at 4° C. and the RNAsedigestion was subsequently carried out at 37° C. for 1 hour. Storage ofthe DNA took place at 4° C.

Example 3

Isolation of Total RNA and Poly-(A)+RNA and cDNA Library Constructionfrom Physcomitrella patens

For the investigation of transcripts, both total RNA and poly-(A)⁺ RNAwere isolated. The total RNA was obtained from wild-type 9 day oldprotonemata following the GTC-method (Reski et al. 1994, Mol. Gen.Genet., 244:352-359). The Poly(A)+ RNA was isolated using Dyna Beads®(Dynal, Oslo, Norway) following the instructions of the manufacturersprotocol. After determination of the concentration of the RNA or of thepoly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

For cDNA library construction, first strand synthesis was achieved usingMurine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany)and oligo-d(T)-primers, second strand synthesis by incubation with DNApolymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours),16° C. (1 hour) and 22° C. (1 hour). The reaction was stopped byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche,Mannheim) at 37° C. (30 minutes). Nucleotides were removed byphenol/chloroform extraction and Sephadex G50 spin columns. EcoRIadapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends byT4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated byincubation with polynucleotide kinase (Roche, 37° C., 30 minutes). Thismixture was subjected to separation on a low melting agarose gel. DNAmolecules larger than 300 base pairs were eluted from the gel, phenolextracted, concentrated on Elutip-D-columns (Schleicher and Schuell,Dassel, Germany) and were ligated to vector arms and packed into lambdaZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit(Stratagene, Amsterdam, Netherlands) using material and following theinstructions of the manufacturer.

Example 4

Sequencing and Function Annotation of Physcomitrella patens ESTs

cDNA libraries as described in Example 3 were used for DNA sequencingaccording to standard methods, and in particular, by the chaintermination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands.Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (see Sambrook et al. 1989 ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNApreparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used:

5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO:16 5′-CTAAAGGGAACAAAAGCTG-3′ SEQ IDNO:17 5′-TGTAAAACGACGGCCAGT-3′ SEQ ID NO:18

Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference the website at pedant.mips.biochem.mpgde. The most importantalgorithms incorporated in EST-MAX are: FASTA: Very sensitive sequencedatabase searches with estimates of statistical significance; Pearson W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol. 183:63-98; BLAST: Very sensitive sequence databasesearches with estimates of statistical significance. Altschul S. F.,Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignmentsearch tool. Journal of Molecular Biology 215:403-10; PREDATOR:High-accuracy secondary structure prediction from single and multiplesequences. Frishman, D. and Argos, P. (1997) 75% accuracy in proteinsecondary structure prediction. Proteins, 27:329-335; CLUSTALW: Multiplesequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994) CLUSTAL W: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research,22:4673-4680; TMAP: Transmembrane region prediction from multiplyaligned sequences. Persson, B. and Argos, P. (1994) Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments. J. Mol. Biol. 237:182-192; ALOM2: Transmembrane regionprediction from single sequences. Klein, P., Kanehisa, M., and DeLisi,C. Prediction of protein function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226(1984). Version 2 by Dr. K. Nakai; PROSEARCH: Detection of PROSITEprotein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M.,Smith J. E. (1992) ProSearch: fast searching of protein sequences withregular expression patterns related to protein structure and function.Biotechniques 13, 919-921; BLIMPS: Similarity searches against adatabase of ungapped blocks. J. C. Wallace and Henikoff S., (1992);PATMAT: A searching and extraction program for sequence, pattern andblock queries and databases, CABIOS 8:249-254. Written by Bill Alford.

Example 5

Identification of Physcomitrella patens ORFS Corresponding to PLC-1,PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1

The Physcomitrella patens partial cDNAs (ESTs) shown in Table 1 belowwere identified in the Physcomitrella patens EST sequencing programusing the program EST-MAX through BLAST analysis. The SequenceIdentification Numbers corresponding to these ESTs are as follows: PLC-1(SEQ ID NO:1); PLC-2 (SEQ ID NO:2); 14-3-3P-1 (SEQ ID NO:3); 14-3-3P-2(SEQ ID NO:4) and CBP-1 (SEQ ID NO:5).

TABLE 1 Functional ORF Name categories Function Sequence code positionPpPLC-1 Signal phosphoinositide-specific c_pp004040301r 1-759transduction phospholipase C PpPLC-2 Signal phosphoinositide-specificc_pp004041126r 2-484 transduction phospholipase C Pp14-3-3P-1 Signal14-3-3 protein c_pp002015016r 1115-555   transduction Pp14-3-3-P-2Signal 14-3-3 brain protein homolog s_pp001097008r 479-216  transductionCBP-1 Signal EF-Hand containing protein- c_pp004076327r 1-566transduction like

TABLE 2 Degree of amino acid identity and similarity of PpPLC-1 andother homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q43442 P93341 Q43439 O49902 Q43443 Protein PHOSPHO-PHOSPHO- PHOSPHA- 1-PHOSPHA- PHOSPHO- name INOSITIDE- INOSITIDE- TIDYLTIDYL- INOSITIDE- SPECIFIC SPECIFIC INOSITOL- INOSITOL- SPECIFICPHOSPHO- PHOSPHO- SPECIFIC 4,5-BISPHOS- PHOSPHO- LIPASE C LIPASE CPHOSPHO- PHATE LIPASE C P12 LIPASE C PHOSPHODI- P13 ESTERASE SpeciesGlycine max Nicotiana Glycine max Nicotiana Glycine (Soybean) rustica(Soybean) rustica max (Aztec (Aztec (Soybean) tobacco) tobacco) Identity% 46% 43% 43% 43% 43% Similarity % 57% 54% 53% 53% 53%

TABLE 3 Degree of amino acid identity and similarity of PpPLC-2 andother homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q43442 O49902 Q43443 P93341 Q43439 Protein PHOSPHO-1-PHOSPHA- PHOSPHO- PHOSPHO- PHOSPHA- name INOSITIDE- TIDYL- INOSITIDE-INOSITIDE- TIDYL- SPECIFIC INOSITOL- SPECIFIC SPECIFIC INOSITOL-PHOSPHO- 4,5- PHOSPHO- PHOSPHO- SPECIFIC LIPASE C BISPHOS- LIPASE CLIPASE C PHOSPHO- P12 PHATE P13 LIPASE C PHOSPHODI- ESTERASE SpeciesGlycine Nicotiana Glycine Nicotiana Glycine max rustica max rustica max(Soybean) (Aztec (Soybean) (Aztec (Soybean) tobacco) tobacco) Identity %44% 41% 42% 40% 42% Similarity % 54% 53% 53% 52% 53%

TABLE 4 Degree of amino acid identity and similarity of Pp14-3-3P-1 andother homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9LKK9 Q9M5W3 Q96453 O49152 P42654 Protein 14-3-3 14-3-3-14-3-3- 14-3-3 14-3-3- name PROTEIN LIKE LIKE PROTEIN LIKE PROTEINPROTEIN HOMOLOG PROTEIN D B Species Populus Euphorbia Glycine MaackiaVicia faba alba × esula max amurensis (Broad Populus (Leafy (Soybean)bean) tremula spurge) Identity 80% 80% 78% 78% 77% % Similar- 90% 88%87% 88% 88% ity %

TABLE 5 Degree of amino acid identity and similarity of Pp14-3-3P-2 andother homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9SP07 P93785 O24222 O49082 Q9XEW8 Protein 14-3-3- 14-3-3GF14-C GF14 14-3-3 name LIKE PROTEIN PROTEIN PROTEIN PROTEIN PROTEINSpecies Lilium Solanum Oryza Fritillaria Picea longiflorum tuberosumsativa agrestis glauca (Trumpet (Potato) (Rice) (White lily) spruce)Identity 79% 80% 80% 79% 80% % Similar- 86% 87% 88% 87% 88% ity %

TABLE 6 Degree of amino acid identity and similarity of PpCBP-1 andother homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9T0I9 O82062 O64866 Q42438 Q9SQI4 Protein EF-HAND 39 KDAEF- F4I1.12 CALCIUM- CENTRIN name CONTAINING HAND PROTEIN DEPENDENTPROTEIN- CONTAINING PROTEIN LIKE PROTEIN KINASE Species ArabidopsisSolanum Arabidopsis Arabidopsis Nicotiana thaliana tuberosum thalianathaliana tabacum (Mouse-ear (Potato) (Mouse-ear (Mouse-ear (Commoncress) cress) cress) tobacco) Identity % 19% 20% 24% 10% 16% Similarity% 34% 35% 41% 21% 28%

Example 6

Cloning of the Full-Length Physcomitrella patens cDNA Encoding forPLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1

To isolate the clones encoding PLC-1, PLC-2, 14-3-3P-2 and CBP-1 fromPhyscomitrella patens, cDNA libraries were created with SMART RACE cDNAAmplification kit (Clontech Laboratories) following manufacturer'sinstructions. Total RNA isolated as described in Example 3 was used asthe template. The cultures were treated prior to RNA isolation asfollows: Salt Stress: 2, 6, 12, 24, 48 hours with 1-M NaCl-supplementedmedium; Cold Stress: 4° C. for the same time points as for salt; DroughtStress: cultures were incubated on dry filter paper for the same timepoints as for salt.

5′ RACE Protocol

The EST sequences PLC-1 (SEQ ID NO:1), PLC-2 (SEQ ID NO:2), 14-3-3P-2(SEQ ID NO:4) and CBP-1 (SEQ ID NO:5) identified from the databasesearch as described in Example 5 were used to design oligos for RACE(see Table 7). The extended sequences for these genes were obtained byperforming Rapid Amplification of cDNA Ends polymerase chain reaction(RACE PCR) using the Advantage 2 PCR kit (Clontech Laboratories) and theSMART RACE cDNA amplification kit (Clontech Laboratories) using aBiometra T3 Thermocycler following the manufacturer's instructions. Thesequences obtained from the RACE reactions corresponded to full-lengthcoding regions of PLC-1, PLC-2, 14-3-3P-2 and CBP-1 were used to designoligos for full-length cloning of the respective genes (see belowfull-length amplification).

Full-length Amplification

Full-length clones corresponding PLC-1 (SEQ ID NO:1); PLC-2 (SEQ IDNO:2); 14-3-3P-1 (SEQ ID NO:3); 14-3-3P-2 (SEQ ID NO:4) and CBP-1 (SEQID NO:5) were obtained by performing polymerase chain reaction (PCR)with gene-specific primers (see Table 7) and the original EST as thetemplate. The conditions for the reaction were standard conditions withPWO DNA polymerase (Roche). PCR was performed according to standardconditions and to manufacturer's protocols (Sambrook et al., 1989Molecular Cloning, A Laboratory Manual. 2nd Edition. Cold Spring HarborLaboratory Press. Cold Spring Harbor, N.Y., Biometra T3 Thermocycler).The parameters for the reaction were: five minutes at 94° C. followed byfive cycles of one minute at 94° C., one minute at 50° C. and 1.5minutes at 72° C. This was followed by twenty five cycles of one minuteat 94° C., one minute at 65° C. and 1.5 minutes at 72° C.

The amplified fragments were extracted from agarose gel with a QIAquickGel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector(Invitrogen) following manufacturer's instructions. Recombinant vectorswere transformed into Top10 cells (Invitrogen) using standard conditions(Sambrook et al. 1989. Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.).Transformed cells were selected for on LB agar containing 100 μg/mlcarbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside)and 0.8 mg IPTG (isopropylthio-β-D-galactoside) grown overnight at 37°C. White colonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al., 1989 Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.).

TABLE 7 Sites in the final Isolation Gene product Method Primers RacePrimer Full-length PCR PpPLC-1 XmaI/HpaI 5′ RACE RC204: RC642: and RT-(SEQ ID NO:19) (SEQ ID NO:20) PCR for CAGGTCCGAGC ATCCCGGGCAATCGTCGFull-length TGACGATGAAC GGTGACATTCCTGTTC clone CCAG RC643: (SEQ IDNO:21) GCGTTAACCAACACCTC AGCGTTCCACATGCAT PpPLC-2 XmaI/HpaI 5′ RACERC203: RC608: and RT- (SEQ ID NO:22) (SEQ ID NO:23) PCR for CGAGCTCCTCCATCCCGGGCTTCGGGAG Full-length ACCAGATTCCT TTTAAGAGGATGTCACG clone GTTCRC609: (SEQ ID NO:24) GCGTTAACCTTGGGTGC ACACACTAAACTGGTC Pp14-3-XmaI/SacI RT-PCR for RC399: 3P-1 Full-length (SEQ ID NO:25) cloneATCCCGGGCGGACTGTC GTGGACGAGTGTGCTAG RC400: (SEQ ID NO:26)GCGAGCTCGGCACGCA ACTGCACATCTTCTTGC Pp14-3- HpaI/SacI 5′ RACE RC195:RC548: 3P-2 and RT- (SEQ ID NO:27) (SEQ ID NO:28) PCR for ACCAGCCTCAAGCGTTAACTTCACAATG Full-length CTTAGTCGCCT ACGGAGCTACGAGAGG clone GGAC ARC549: (SEQ ID NO:29) GCGAGCTCCAGCCTCAA CTTAGTCGCCTGGACA PpCBP-1XmaI/SacI 5′ RACE RC197: RC654: and RT- (SEQ ID NO:30) (SEQ ID NO:31)PCR for CCCTGCTCAAC ATCCCGGGTCAGCTCGT Full-length GCCCAGCTGCAGGAAGTGTTGCAGCA clone TAAT RC655: (SEQ ID NO:32) GCGAGCTCGTCCAATTTTCACTCGGGGGCTTCC

Example 7

Engineering Stress-tolerant Arabidopsis Plants by Over-expressing theGenes PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1

Binary Vector Construction: pACGH101

The plasmid construct pACGH101 was digested with PstI (Roche) and FseI(NEB) according to manufacturers' instructions. The fragment waspurified by agarose gel and extracted via the Qiaex II DNA Extractionkit (Qiagen). This resulted in a vector fragment with the ArabidopsisActin2 promoter with internal intron and the OCS3 terminator. Primersfor PCR amplification of the NPTII gene were designed as follows:

5′NPT-Pst: (SEQ ID NO:33) GCG-CTG-CAG-ATT-TCA-TTT-GGA-GAG-GAC-ACG3′NPT-Fse: (SEQ ID NO:34)CGC-GGC-CGG-CCT-CAG-AAG-AAC-TCG-TCA-AGA-AGG-CG.

The 0.9 kilobase NPTII gene was amplified via PCR from pCambia 2301plasmid DNA [94° C. 60 sec, {94° C. 60 sec, 61° C. (−0.1° C. per cycle)60 sec, 72° C. 2 min}×25 cycles, 72° C. 10 min on Biometra T-Gradientmachine], and purified via the Qiaquick PCR Extraction kit (Qiagen) asper manufacturer's instructions. The PCR DNA was then subcloned into thepCR-BluntII TOPO vector (Invitrogen) pursuant to the manufacturer'sinstructions (NPT-Topo construct). These ligations were transformed intoTop10 cells (Invitrogen) and grown on LB plates with 50 ug/ml kanamycinsulfate overnight at 37° C. Colonies were then used to inoculate 2 ml LBmedia with 50 ug/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen)and sequenced in both the 5′ and 3′ directions using standardconditions. Subsequent analysis of the sequence data using VectorNTIsoftware revealed no PCR errors present in the NPTII gene sequence.

The NPT-Topo construct was then digested with PstI (Roche) and FseI(NEB) according to manufacturers' instructions. The 0.9 kilobasefragment was purified on agarose gel and extracted by Qiaex II DNAExtraction kit (Qiagen). The Pst/Fse insert fragment from NPT-Topo andthe Pst/Fse vector fragment from pACGH101 were then ligated togetherusing T4 DNA Ligase (Roche) following manufacturer's instructions. Theligation was then transformed into Top10 cells (Invitrogen) understandard conditions, creating pBPSsc019 construct. Colonies wereselected on LB plates with 50 ug/ml kanamycin sulfate and grownovernight at 37° C. These colonies were then used to inoculate 2 ml LBmedia with 50 ug/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen)following the manufacturer's instructions.

The pBPSSC019 construct was digested with KpnI and BsaI (Roche)according to manufacturer's instructions. The fragment was purified viaagarose gel and then extracted via the Qiaex II DNA Extraction kit(Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPTcassette, which included the Arabidopsis Actin2 promoter with internalintron, the NPTII gene and the OCS3 terminator.

The pBPSJH001 vector was digested with SpeI and ApaI (Roche) andblunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) accordingto manufacture's instructions. This produced a 10.1 kilobase vectorfragment minus the Gentamycin cassette, which was recircularized byself-ligating with T4 DNA Ligase (Roche), and transformed into Top10cells (Invitrogen) via standard conditions. Transformed cells wereselected for on LB agar containing 50 μg/ml kanmycin sulfate and grownovernight at 37° C. Colonies were then used to inoculate 2 ml of liquidLB containing 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen)following manufacture's instructions. The recircularized plasmid wasthen digested with KpnI (Roche) and extracted from agarose gel via theQiaex II DNA Extraction kit (Qiagen) as per manufacturers' instructions.

The Act-NPT Kpn-cut insert and the Kpn-cut pBPSJH001 recircularizedvector were then ligated together using T4 DNA Ligase (Roche) andtransformed into Top10 cells (Invitrogen) as per manufacturers'instructions. The resulting construct, pBPSsc022, now contained theSuper Promoter, the GUS gene, the NOS terminator, and the Act-NPTcassette. Transformed cells were selected for on LB agar containing 50μg/ml kanmycin sulfate and grown overnight at 37° C. Colonies were thenused to inoculate 2 ml of liquid LB containing 50 μg/ml kanamycinsulfate and grown overnight at 37° C. Plasmid DNA was extracted usingthe QIAprep Spin Miniprep Kit (Qiagen) following manufacturer'sinstructions. After confirmation of ligation success via restrictiondigests, pBPSsc022 plasmid DNA was further propagated and recoveredusing the Plasmid Midiprep Kit (Qiagen) following the manufacturer'sinstructions.

Subcloning of PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 into theBinary Vector

The fragments containing the different Physcomitrella patens signaltransduction factors were subcloned from the recombinant PCR2.1 TOPOvectors by double digestion with restriction enzymes (see Table 8)according to manufacturer's instructions. The subsequence fragment wasexcised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen)according to manufacturer's instructions and ligated into the binaryvector pBPSsc022, cleaved with appropriate enzymes (see Table 8) anddephosphorylated prior to ligation. The resulting recombinant pBPSsc022vector contained the corresponding transcription factor in the senseorientation under the control of the constitutive super promoter.

TABLE 8 Listed are the names of the various constructs of thePhyscomitrella patens transcription factors used for planttransformation Enzymes used to generate gene Enzymes used to BinaryVector Gene fragment restrict pBPSJH001 Construct PpPLC-1 XmaI/HpaIXmaI/Ecl136 pBPSSY009 PpPLC-2 XmaI/HpaI XmaI/Ecl136 pBPSJYW009Pp14-3-3P-1 XmaI/SacI SmaI/SacI pBPSJYW018 Pp14-3-3P-2 HpaI/SacISmaI/SacI pBPSSY031 PpCBP-1 XmaI/SacI XmaI/SacI PBPSLVM182Agrobacterium Transformation

The recombinant vectors were transformed into Agrobacterium tumefaciensC58C1 and PMP90 according to standard conditions (Hoefgen andWillmitzer, 1990).

Plant Transformation

Arabidopsis thaliana ecotype C24 were grown and transformed according tostandard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1194-1199;Bent et al. 1994, Science 265:1856-1860).

Screening of Transformed Plants

T1 seeds were sterilized according to standard protocols (Xiong et al.1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were platedon ½ Murashige and Skoog media (MS) (Sigma-Aldrich) pH 5.7 with KOH,0.6% agar and supplemented with 1% sucrose, 0.5 g/L2-[N-Morpholino]ethansulfonic acid (MES) (Sigma-Aldrich), 50 μg/mlkanamycin (Sigma-Aldrich), 500 μg/ml carbenicillan (Sigma-Aldrich) and 2μg/ml benomyl (Sigma-Aldrich). Seeds on plates were vernalized for fourdays at 4° C. The seeds were germinated in a climatic chamber at an airtemperature of 22° C. and light intensity of 40 micromol s⁻¹m⁻² (whitelight; Philips TL 65W/25 fluorescent tube) and 16 hours light and 8hours dark day length cycle. Transformed seedlings were selected after14 days and transferred to ½ MS media pH 5.7 with KOH 0.6% agar platessupplemented with 0.6% agar, 1% sucrose, 0.5 g/L MES (Sigma-Aldrich),and 2 μg/ml benomyl (Sigma-Aldrich) and allowed to recover forfive-seven days.

Drought Tolerance Screening

T1 seedlings were transferred to dry, sterile filter paper in a petridish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Percieval Growth Cabinet MLR-350H, micromole s⁻¹m⁻²(white light; Philips TL 65W/25 fluorescent tube). The RH was thendecreased to 60% and the seedlings were desiccated further for eighthours. Seedlings were then removed and placed on ½ MS 0.6% agar platessupplemented with 2 μg/ml benomyl (Sigma-Aldrich) and 0.5 g/L MES(Sigma-Aldrich) and scored after five days.

Under drought stress conditions, PpPLC-1 over-expressing Arabidopsisthaliana plants showed a 70% (7 survivors from 10 stressed plants)survival rate to the stress screening; PpPLC-2, 98% (50 survivors from51 stressed plants); Pp-14-3-3P-1, 80% (12 survivors from 15 stressedplants); Pp-14-3-3P-2, 100% (22 survivors from 22 stressed plants); andPpCBP-1, 100% (52 survivors from 52 stressed plants), whereas theuntransformed control only showed a 28% survival rate. It is noteworthythat the analyses of these transgenic lines were performed with T1plants, and therefore, the results will be better when a homozygous,strong expresser is found.

TABLE 9 Summary of the drought stress tests Drought Stress Test Numberof Total number of Percentage of Gene Name survivors plants survivorsPpPLC-1 7 10 70% PpPLC-2 50 51 98% Pp14-3-3P-1 12 15 80% Pp14-3-3P-2 2222 100% PpCBP-1 52 52 100% Control 16 57 28%Freezing Tolerance Screening

Seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings were incubated at 4° C. for 1 hour and then covered withshaved ice. The seedlings were then placed in an EnvironmentalSpecialist ES2000 Environmental Chamber and incubated for 3.5 hoursbeginning at −1.0° C. decreasing 1° C./hour. The seedlings were thenincubated at −5.0° C. for 24 hours and then allowed to thaw at 5° C. for12 hours. The water was poured off and the seedlings were scored after 5days.

Under freezing stress conditions, PpPLC-2 over-expressing Arabidopsisthaliana plants showed a 78% (18 survivors from 23 stressed plants)survival rate; Pp-14-3-3P-1, 100% (6 survivor from 6 stressed plants)and PpCBP-1, 78% (18 survivor from 23 stressed plants), whereas theuntransformed control only showed a 2% (1 survivors from 48 testedplants) survival rate. It is noteworthy that the analyses of thesetransgenic lines were performed with T1 plants, and therefore, theresults will be better when a homozygous, strong expresser is found.

TABLE 10 Summary of the freezing stress tests Freezing Stress Test Totalnumber of Percentage of Gene Name Number of survivors plants survivorsPpPLC-2 18 23 78% Pp14-3-3P-1 6 6 100% PpCBP-1 25 37 68% Control 1 48 2%Salt Tolerance Screening

Seedlings were transferred to filter paper soaked in ½ MS and placed on½ MS 0.6% agar supplemented with 2 μg/ml benomyl the night before thesalt tolerance screening. For the salt tolerance screening, the filterpaper with the seedlings was moved to stacks of sterile filter paper,soaked in 50 mM NaCl, in a petri dish. After two hours, the filter paperwith the seedlings was moved to stacks of sterile filter paper, soakedwith 200 mM NaCl, in a petri dish. After two hours, the filter paperwith the seedlings was moved to stacks of sterile filter paper, soakedin 600 mM NaCl, in a petri dish. After 10 hours, the seedlings weremoved to petri dishes containing ½ MS 0.6% agar supplemented with 2μg/ml benomyl. The seedlings were scored after 5 days.

The transgenic plants are screened for their improved salt tolerancedemonstrating that transgene expression confers salt tolerance.

Example 8

Detection of the PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 Transgenesin the Transgenic Arabidopsis Lines

To check for the presence of the PpPLC-1, PpPLC-2, Pp-14-3-3P-1,Pp-14-3-3P-2 and PpCBP-1 transgenes in transgenic Arabidopsis lines, PCRwas performed on genomic DNA which contaminates the RNA samples taken asdescribed in Example 9 below. 2.5 μl of RNA sample was used in a 50 μlPCR reaction using Taq DNA polymerase (Roche Molecular Biochemicals)according to the manufacturer's instructions. The primer for the binaryvector region (5′GCTGACACGCCAAGCCTCGCTAGTC3′) (SEQ ID NO:35) and thegene specific 3′ primer for each transgene which was used for thefull-length RT-PCR (see Table 7) were used for the PCR. The PCR programwas as following: 30 cycles of 1 minute at 94° C., 1 minute at 62° C.and 4 minutes at 70° C., followed by 10 minutes at 72° C. Binary vectorplasmid with the transgenes cloned in was used as positive control, andthe wild type C24 genomic DNA was used as negative control in the PCRreactions. 10 μl PCR reaction was analyzed on 0.8% agarose—ethidiumbromide gel.

The transgenes with the expected size (for PpPLC-1: 2.3 kb fragment;PpPLC-2: 2.2 kb fragment; Pp-14-3-3P-1: 0.9 kb fragment; Pp-14-3-3P-2:0.9 kb fragment; PpCBP -1: 1,8 kb fragment) were successfully amplifiedfrom the T1 transgenic lines and positive control, but not from thewild-type C24. This result indicates that the T1 transgenic plantscontain at least one copy of the transgenes. There was no indication ofexistence of either identical or very similar in untransformedArabidopsis thaliana which can be amplified in this method in thewild-type plants.

Example 9

Detection of the PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 TransgenemRNA in Transgenic Arabidopsis Lines

Transgene expression was detected using RT-PCR. Total RNA was isolatedfrom stress-treated plants using a procedure adapted from (Verwoerd etal., 1989 NAR 17:2362). Leaf samples (50-100 mg) were collected andground to a fine powder in liquid nitrogen. Ground tissue wasresuspended in 500 μl of a 80° C., 1:1 mixture, of phenol to extractionbuffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS), followed bybrief vortexing to mix. After the addition of 250 μl of chloroform, eachsample was vortexed briefly. Samples were then centrifuged for 5 minutesat 12,000×g. The upper aqueous phase was removed to a fresh eppendorftube. RNA was precipitated by adding 1/10^(th) volume 3M sodium acetateand 2 volumes 95% ethanol. Samples were mixed by inversion and placed onice for 30 minutes. RNA was pelleted by centrifugation at 12,000×g for10 minutes. The supernatant was removed and pellets briefly air-dried.RNA sample pellets were resuspended in 10 μl DEPC treated water. Toremove contaminating DNA from the samples, each was treated withRNase-free DNase (Roche) according to the manufacturer'srecommendations. cDNA was synthesized from total RNA using the 1^(st)Strand cDNA synthesis kit (Boehringer Mannheim) following manufacturer'srecommendations.

PCR amplification of a gene-specific fragment from the synthesized cDNAwas performed using Taq DNA polymerase (Roche) and gene-specific primersas shown below in the following reaction: 1×PCR buffer, 1.5 mM MgCl₂,0.2 μM each primer, 0.2 μM dNTPs, 1 unit polymerase, 5 μl cDNA fromsynthesis reaction. Amplification was performed under the followingconditions: Predenaturation, 94° C., 3 minutes; denaturation, 94° C., 30seconds; annealing, 62° C., 30 seconds; extension, 72° C., 2 minute, 30cycles; extension, 72° C., 5 minutes; hold, 4° C., forever. PCR productswere run on a 1% agarose gel, stained with ethidium bromide, andvisualized under UV light using the Quantity-One gel documentationsystem (Bio-Rad).

Expression of the transgenes was detected in the T1 transgenic line.These results indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic lines. In agreement with theprevious statement, no expression of identical or very similarendogenous genes could be detected by this method. These results are inagreement with the data from Example 7.

TABLE 11 Primers used for the amplification of respective transgene mRNAin PCR using RNA isolated from transgenic Arabidopsis thaliana plants astemplate Gene 5′ primer 3′ primer PpPLC-1 5′CCAGCTTAGCAGCGACA5′CAGTCTGTCTTCCACGG GTAGCGACGT3′ TAGTTCCT3′ (SEQ ID NO:36) (SEQ IDNO:37) PpPLC-2 5′GGCCATGGAGAACAGGA 5′GCTCCGTAGTTCCAAGC ATCTGGTGG3′CAGAGTAG3′ (SEQ ID NO:38) (SEQ TD NO:39) Pp14-3-3P-1 Same primers usedSame primers used for the full length for the full length RT-PCR RT-PCR(see Table 1) (see Table 1) Pp14-3-3P-2 Same primers used Same primersused for the full length for the full length RT-PCR RT-PCR (see Table 1)(see Table 1) PpCBP-1 5′GACACTGATGAGAGTG 5′GACTCGATGCTTCAACGGCAAGCTGAG3′ AGAGGCAG3′ (SEQ ID NO:40) (SEQ ID NO:41)

Example 10

Engineering Stress-tolerant Soybean Plants by Over-expressing the PLC-1,PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 Gene

The constructs pBPSSY009, pBPSJYW009, pBPSJYW018, pBPSSY031 andpBPSLVM182 are used to transform soybean as described below.

Seeds of soybean are surface sterilized with 70% ethanol for 4 minutesat room temperature with continuous shaking, followed by 20% (v/v)Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds are rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats are peeled off, andcotyledons are detached from the embryo axis. The embryo axis isexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes are collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LBsolid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin,50 mg/l kanamycin) followed by growth of the single colony in liquid LBmedium to an optical density at 600 nm of 0.8. Then, the bacteriaculture is pelleted at 7000 rpm for 7 minutes at room temperature, andresuspended in MS (Murashige and Skoog, 1962) medium supplemented with100 μM acetosyringone. Bacteria cultures are incubated in thispre-induction medium for 2 hours at room temperature before use. Theaxis of soybean zygotic seed embryos at approximately 15% moisturecontent are imbibed for 2 hours at room temperature with the pre-inducedAgrobacterium suspension culture. The embryos are removed from theimbibition culture and are transferred to Petri dishes containing solidMS medium supplemented with 2% sucrose and incubated for 2 days, in thedark at room temperature. Alternatively, the embryos are placed on topof moistened (liquid MS medium) sterile filter paper in a Petri dish andincubated under the same conditions described above. After this period,the embryos are transferred to either solid or liquid MS mediumsupplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to killthe agrobacteria. The liquid medium is used to moisten the sterilefilter paper. The embryos are incubated during 4 weeks at 25° C., under150 μmol m⁻² sec⁻¹ and 12 hours photoperiod. Once the seedlings produceroots, they are transferred to sterile metromix soil. The medium of thein vitro plants is washed off before transferring the plants to soil.The plants are kept under a plastic cover for 1 week to favor theacclimatization process. Then the plants are transferred to a growthroom where they are incubated at 25° C., under 150 μmol m⁻² sec⁻¹ lightintensity and 12 hours photoperiod for about 80 days.

The transgenic plants are then screened for their improved drought, saltand/or cold tolerance according to the screening method described inExample 7 to demonstrate that transgene expression confers stresstolerance.

Example 11

Engineering Stress-tolerant Rapeseed/Canola Plants by Over-expressingthe PLC-1, PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 Genes

The constructs pBPSSY009, pBPSJYW009, pBPSJYW018, pBPSSY031 andpBPSLVM182 are used to transform rapeseed/canola as described below.

The method of plant transformation described herein is also applicableto Brassica and other crops. Seeds of canola are surface sterilized with70% ethanol for 4 minutes at room temperature with continuous shaking,followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20minutes, at room temperature with continuous shaking. Then, the seedsare rinsed 4 times with distilled water and placed on moistened sterilefilter paper in a Petri dish at room temperature for 18 hours. Then theseed coats are removed and the seeds are air dried overnight in ahalf-open sterile Petri dish. During this period, the seeds lose approx.85% of its water content. The seeds are then stored at room temperaturein a sealed Petri dish until further use. DNA constructs and embryoimbibition are as described in Example 10. Samples of the primarytransgenic plants (TO) are analyzed by PCR to confirm the presence ofT-DNA. These results are confirmed by Southern hybridization in whichDNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

The transgenic plants are then screened for their improved stresstolerance according to the screening method described in Example 7 todemonstrate that transgene expression confers drought tolerance.

Example 12

Engineering Stress-tolerant Corn Plants by Over-expressing the PLC-1,PLC-2, 14-3-3P-1, 14-3-3P-2 or CBP-1 Genes

The constructs pBPSSY009, pBPSJYW009, pBPSJYW018, pBPSSY031 andpBPSLVM182 are used to transform corn as described below.

Transformation of maize (Zea Mays L.) is performed with the methoddescribed by Ishida et al. 1996. Nature Biotch 14745-50. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. This procedure provides a transformation efficiency ofbetween 2.5% and 20%. The transgenic plants are then screened for theirimproved drought, salt and/or cold tolerance according to the screeningmethod described in Example 7 to demonstrate that transgene expressionconfers stress tolerance.

Example 13

Engineering Stress-tolerant Wheat Plants by Over-expressing the PLC-1,PLC-2, 14-3-3P-1, 14-3-3P-2 and CBP-1 Genes

The constructs pBPSSY009, pBPSJYW009, pBPSJYW018, pBPSSY031 andpBPSLVM182 are used to transform wheat as described below.

Transformation of wheat is performed with the method described by Ishidaet al. 1996 Nature Biotch. 14745-50. Immature embryos are co-cultivatedwith Agrobacterium tumefaciens that carry “super binary” vectors, andtransgenic plants are recovered through organogenesis. This procedureprovides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7 demonstratingthat transgene expression confers drought tolerance.

Example 14

Identification of Homologous and Heterologous Genes

Gene sequences can be used to identify homologous or heterologous genesfrom cDNA or genomic libraries. Homologous genes (e.g. full-length cDNAclones) can be isolated via nucleic acid hybridization using for examplecDNA libraries. Depending on the abundance of the gene of interest,100,000 up to 1,000,000 recombinant bacteriophages are plated andtransferred to nylon membranes. After denaturation with alkali, DNA isimmobilized on the membrane by e.g. UV cross linking. Hybridization iscarried out at high stringency conditions. In aqueous solutionhybridization and washing is performed at an ionic strength of 1 M NaCland a temperature of 68° C. Hybridization probes are generated by e.g.radioactive (³²P) nick transcription labeling (High Prime, Roche,Mannheim, Germany). Signals are detected by autoradiography.

Partially homologous or heterologous genes that are related but notidentical can be identified in a manner analogous to the above-describedprocedure using low stringency hybridization and washing conditions. Foraqueous hybridization, the ionic strength is normally kept at 1 M NaClwhile the temperature is progressively lowered from 68 to 42° C.

Isolation of gene sequences with homologies (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radio labeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

-   Oligonucleotide hybridization solution:-   6×SSC-   0.01 M sodium phosphate-   1 mM EDTA (pH 8)-   0.5% SDS-   100 μg/ml denatured salmon sperm DNA-   0.1% nonfat dried milk

During hybridization, temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide Tm or down to room temperature followed bywashing steps and autoradiography. Washing is performed with lowstringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al. (1989), “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.

Example 15

Identification of Homologous Genes by Screening Expression Librarieswith Antibodies

cDNA clones can be used to produce recombinant protein for example in E.coli (e.g. Qiagen QIAexpress pQE system). Recombinant proteins are thennormally affinity purified via Ni-NTA affinity chromatography (Qiagen).Recombinant proteins are then used to produce specific antibodies forexample by using standard techniques for rabbit immunization. Antibodiesare affinity purified using a Ni-NTA column saturated with therecombinant antigen as described by Gu et al., 1994 BioTechniques17:257-262. The antibody can than be used to screen expression cDNAlibraries to identify homologous or heterologous genes via animmunological screening (Sambrook, J. et al. (1989), “Molecular Cloning:A Laboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons).

Example 16

In vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by passage ofplasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M. (1994)Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 17

In vitro Analysis of the Function of Physcomitrella Genes in TransgenicOrganisms

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M., and Webb, E. C.,(1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure andMechanism. Freeman: New York; Walsh, (1979) Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed. (1983) The Enzymes, 3^(rd) ed. Academic Press: New York; Bisswanger,H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds. (1983-1986) Methods ofEnzymatic Analysis, 3^(rd) ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes.VCH: Weinheim, p. 352-363.

The activity of proteins which bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such proteins on the expression ofother molecules can be measured using reporter gene assays (such as thatdescribed in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 andreferences cited therein). Reporter gene test systems are well known andestablished for applications in both pro- and eukaryotic cells, usingenzymes such as β-galactosidase, green fluorescent protein, and severalothers.

The determination of activity of membrane-transport proteins can beperformed according to techniques such as those described in Gennis, R.B. Pores, Channels and Transporters, in Biomembranes, MolecularStructure and Function, pp. 85-137, 199-234 and 270-322, Springer:Heidelberg (1989).

Example 18

Purification of the Desired Product from Transformed Organisms

Recovery of the desired product from plant material (i.e.,Physcomitrella patentsor Arabidopsis thaliana), fungi, algae, ciliates,C. glutamicum cells, or other bacterial cells transformed with thenucleic acid sequences described herein, or the supernatant of theabove-described cultures can be performed by various methods well knownin the art. If the desired product is not secreted from the cells, canbe harvested from the culture by low-speed centrifugation, the cells canbe lysed by standard techniques, such as mechanical force orsonification. Organs of plants can be separated mechanically from othertissue or organs. Following homogenization cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and the supernatefraction is retained for further purification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. Oneskilled in the art would be well-versed in the selection of appropriatechromatography resins and in their most efficacious application for aparticular molecule to be purified. The purified product may beconcentrated by filtration or ultrafiltration, and stored at atemperature at which the stability of the product is maximized.

There is a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York(1986). Additionally, the identity and purity of the isolated compoundsmay be assessed by techniques standard in the art. These includehigh-performance liquid chromatography (HPLC), spectroscopic methods,staining methods, thin layer chromatography, NIRS, enzymatic assay, ormicrobiologically. Such analysis methods are reviewed in: Patek et al.,1994 Appl. Environ. Microbiol. 60:133-140; Malakhova et al., 1996Biotekhnologiya 11:27-32; and Schmidt et al., 1998 Bioprocess Engineer.19:67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol.A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566,575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlasof Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.et al. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

APPENDIX Nucleotide sequence of the partial PLC-1 from Physcomitrellapatens (SEQ ID NO:1) GCACGAGTCCAAGAAGGGGGATTTGGCGCAGGATCTATTGGGGGATGTGTTCTCGACTTACAGCGAGAATGGGAAGCTGGACGCCGAGGGGTTGCTGAAGTTCTTGCAGACAGAGCAAGGGGATAGCAAGTCCTCTCTAGATGACGCCAAGCACCTAGTGGAGTTGATTCGGAATGAGAGACATAAGTCGAAATTCCCTGGGTTCATCGTCAGCTCGGACCTGTCGAAGGGTGATTTTAAAAACTATGTACTGAGCCCGGATTTGAATGGGGTTCTTGAAAGCACTGTGCATCAAGACATGACGCAGCCGTTATCGCACTACTTCATATTCACTGGTCACAACTCGTACTTGACGGGTAACCAGCTTAGCAGCGACAGTAGCGACGTTCCCATTGCTGCTGCACTGCAACGTGGCGTGCGGGTGGTGGAACTGGATTTGTGGCCTGACGATAAAGGCGGCATCAAGGTCACTCACGGGAACACACTCACCAGTCCAGTTGCTTTCGAGAAGTGCATAAAAGCCATCAAGGCCAACGCGTTCGTCTCCTCGAAATATCCTGTAGTTATCACTCTTGAGGATCATCTTTCAAGTCCTTTACAGGCCCTTGCTGCAGAGACTTTGACGAACATTTTGGGAGAGGACTTGTACTATCCACCCTCATCCGATGGGTTTAAAGAACTGCCTTCTCCGGAATCATTGAAAGGGAAAATTCTAATATCTACCAAACCGCCGAAAGAATACCTTGAAGC CGCTGTCGCA Nucleotidesequence of the partial PLC2 from Physcomitrella patens (SEQ ID NO:2)CGGCACCAGGCGGCATGAAGGTCACACACGGAAACACACTTACCAATCCGGTGTCGTTCCAAAAGTGTGTCACAGCCATCAAGAATAACGCCTTCTTCACCTCGGAGTACCCAGTTTGCGTTACTATTGAGGATCATCTTACAAGCGAATTACAGGGCCATGCTGCAGAGATTTTAGAGCAAATTCTCGGAGACGCCCTGTATTATCCACCCACAACTGATGCATTAGTGGAGTTTCCTTCACCGGAGTCACTGAAGAGGAAGATCATAATCTCCACCAAACCGCCGAAGGAGTATCTCGAAGCATGTTCCACGCAGAAATTGGCCATGGAGAACAGGAATCTGGTGGAGGAGCTCGAGAAGGAAGACAAATTGGAGCAGACCACATTCGCTCCCCTTGAAGAGAACCACATCCTGGGAGAAAATACACCATCGCTGCGTAAGGAAGTCGAGGTTTTAAGCCAAAAGGAAATGTCAACACCAAGCTGAGCTTAACTCTAGAAAGTCCCTCCTGGACCTCGGGGAAGCAACCATCCACAAGGTATAGCAAAGAGCAACGATGGCAATGACAACCCTAAAACATTTCAAGTATGCCCGGTTCATCAACATCCGGCTAGCAAACACGCAACGGGGACATCGTGGCGCTCGCACTGCCAGTCGATGGATCAGAACGGATCAGCGGCGATCNATGAAAAGGGGAA TGCCGA Nucleotidesequence of the partial 14-3-3P-1 from Physcomitrella patens (SEQ IDNO:3) TTTTTTTTTAATTGTAAACCGCACAGCCCGGACACAAAACCATCCCACTGCACGAGTCAGATATAACCGTACATGATGTTTATAATGCATTGTTCATTTTAATATTAAAATCTTAGCATTCCTCATCCTAGCCAACGAAGGAAAGAAAAAAGAGAAAAGAAAAGGACAAAAGAGGAAATCTATGAACAGCCAGTCTCTACGCAGAACAAAATAGGCAAGACATGTCATACAACCGCCAGCACGGAACTACACTACGTCTCGCCCCGGCCTACCACCAATCAATTGAGGCTCTTTCCGAATCCACCCCATCAGAAAAGACTTGATTGTCGTGATCACTCCGCTTCCTCCGGTCTCATGTCATCTCCCTTTCCCTGGTCGTCGCCTACATCGTCCTGGAGATCTGACGTCCATAAAGTCAGATTATCTCTAAGTAGTTGCATGATCAATGTGCTGTCCTTGTACGACTCCTCACTCAATGTGTCCAATTCGGCAATTGCCTCGTCGAATGCTTGCTTCGCCAAATGGCATGCCCGCTCAGGGGAGTTCAAAATCTCATAATAGAAGACAGAGAAGTTCAAGGCCAGTCCCAGCCGAATCGGATGAGTTGGCGCCAAGTCTGTCACTTGCTGTATTAGATGCAGCCTGGTAGGCCTTCAAAGATTGGTCAGCAGCTTCTTTTCTCTCAGCCCCAGTTTTGAACTCCGCCAGGTACCGATAGTAATCTCCCTTCATTTTATAGTAGAACACAGTGGACTCTCCCGTGCTGGACGAAGGAATCAAATGTCCGTCGATGATAGACAGGATATCATTGCAGATCTTCGACAGCTCCTCCTCCACCTTGTGTCTGTAGTCCTTGATGCGTTTAACATTCTGTTCGTTACCTTTGCTCTCCTCCTTCTGTTCGATGGATGACATGATCCGCCATGACGCCCTCCGGGCTCCGATGACATTCTTATAACCCACGGACAAGAGATTTCGCTCCTCTACTGTCAGCTCCACATCAAGCTTGGCAACCTTCTTCATCGATTCCACCATCTCATCGTAACGCTCCGCCTGCTCGGCGAGCTTGGCCATGTACACATAGCTCTCGCGCTCCTTCTCCGTACTCATCTTGGCCTAGCACACTCGTCCACGACAGTCCGAAATAGCAGTATCGCGCACCGTCCCCCGAGCACAAACCAAGCGCAGAACGGCAACACACTATGCAATGTAAAGGAAACGCAGACACAAGAGGAGACGGAAAAAACAATAGAGGCAGGAAGAGAGTGGGAGAGAAGAGACGGGGGAGCGGGGCGATGGA GGAGCACGGTGAGCTGGTGCNucleotide sequence of the partial 14-3-3P-2 from Physcomitrella patens(SEQ ID NO:4) GCACGAGCACTGTTACATCGTCGTAGATCTGGTCAGATACCANAACCGGCGAGAAGCATGCAACACAAGAAAACCTGGAGATGTAATGGTTATGCCGATAGGGTTTCATTTAAAACAATCTACATAACCCAGTGCTAATTGTTCTGGGAAGTCGAGCACATATCCGTACCAGCCTCAACTTAGTCGCCTGGACATGAGTTTCTATTCTAAGTTCTAGTGGTCATCANCATCTTCGACCTTGGAATCCTTTCCTTCTTCACCAATGNCGTCCTGCATATCTGAAGTCCATAAGGTAAGGTTATCCCGGAGCAGCTGCATAATGAGAGTACTGTCTTTGTAGGATTCCTCTCCTAAGGTATCTAACTCAGAGATAGCTTCATCAAAAGCCTGCTTGGCAAGATGGCATGCTCGGTCTGGAGAAACCAAAATTTCGTAGTAAAAGACAGAAAAATTCAAAGCCAATCCCAATCTGATCTCGTGCC Nucleotide sequence of the partialCBP-1 from Physcomitrella patens (SEQ ID NO:5)GCACCAGGTTTTAGTAACAGATGGGAAATACTGCAAGAGCGTTTGGTTGTTGAAAGGGCCTACAGTTGTAAACTAGGCGATCACAAGTGCATTGCTTCATTGAGCTTTCGCATGGACTCAATCCAGCTATTGATCTCAATCTTTGTTTTTTCTAAAACATTTTTTCACACAGATGAGCCGGTAGTAGTGTCAGTACTAGACGGCTCAGCAATTAAGGCTTTGCTAGAAGATGAAGATGATTTTGCGATGGTTGCCGAGGATCTGTTTGAGAAGTTAGACACTGATGAGAGTGGCAAGCTGAGCAGCAAAGAGCTTCGACCTGCCATTATGCAGCTGGGCGTTGAGCAGGGTGTCCCTCCTGCCGCAGCTACTACTGAAGCGGAGGAATTGGTTACCAAGTTGATCAACAAGTACGGCCAGGGAACCGAGGAGCTTGGACAAGCTCAATTTGCTGCATTATTGCAAGATGTCCTTCAGGATATGGCCGAGTCTCTTGCAGAGAAACCTATCACAATTGTACGAGATGTGAAAATGCTCAACGGCTCTCATTTGCGAAAGATGCTTGGCTGATGAGAAGGCATTCAAAGGAAATGGCAGATAACATGTTTTAATTGACCTCAGATGTCAACAAAAGATCAACGCTTGAGCAAGTGAAATCAGACCATTATTTGAACAACAAACTGAGCGTGGGGGTCTACCTCCCGTGGTGATTCGGCACAAANAATATTGACGAGTTTCAGGCGTGNTCCACAAACATGGGNAGTGAAGCCGNTTTGGATCGGCANACCCCGCGGTTTGGG AACNNGGGGTCNucleotide sequence of the full-length PLC-1 from Physcomitrella patens(SEQ ID NO:6) ATCCCGGGAATCGTCGGGTGACATTCCTGTTCCTACTGCTGTTTGGCCCATTCGTCCACTCGGCCCATCTCCACTGACTTCCCCAATTGAGATCAGATGGTCTTGTAAGGCAGAGAGCGAGCGAGAGAGAGAGAGAGAGAGAGAGAGAGATTGGGAGTTGAGGCGAAGGGGAGGTGTCGGAGGGGATTTATTGTGCCGTAGCTGGGTTTGCAAAAATGTGTTCTATTCCGTTCGGTCGGAAGAAGTCCAAGAAGGGGGATTTGGCGCAGGATCTGTTGGGGGATGTGTTCTCGACTTACAGCGAGAATGGGAAGCTGGACGCCGAGGGGTTGCTGAAGTTCTTGCAGACAGAGCAAGGGGATAGCAAGTCCTCTCTAGATGACGCCAAGCATTTAGTGGAGTTGATTCGGAATGAGAGACATAAGTCGAAATTCCCTGGGTTCATCGTCAGCTCGGACCTGTCGAAGGGTGATTTTAAAAACTATGTACTGAGCCCGGATTTGAATGGGGTTCTTGAAAGCACTGTGCATCAAGACATGACGCAGCCGTTATCGCACTACTTCATATTCACTGGTCACAACTCGTACTTGACGGGTAACCAGCTTAGCAGCGACAGTAGCGACGTTCCCATTGCTGCTGCACTGCAACGTGGCGTGCGGGTGGTGGAACTGGATTTGTGGCCTGACGATAAAGGCGGCATCAAGGTCACTCACGGGAACACACTCACCAGTCCAGTTGCTTTCGAGAAGTGCATAAAAGCCATCAAGGCCAACGCGTTCGTCTCCTCGAAATATCCTGTAGTTATCACTCTTGAGGATCATCTTTCAAGTCCTTTACAGGCCCTTGCTGCAGAGACTTTGACGAACATTTTGGGAGAGGACTTGTACTATCCACCCTCATCCGATGGGTTTAAAGAACTGCCTTCTCCGGAATCATTGAAAGGGAAAATTCTAATATCTACCAAACCGCCGAAAGAATACCTTGAAGCCGCTGTCGCACAGAAGTCGGCGTTGAAAGATGAAAAGATTTTGAATGAGTTCAAGAAGGCAGATAAGTTGCAGGAGCAGTCAACTGCTCCTGTTAAAAGCCCCGTTGAGAAAAAGATTGCAGTTCCACCATCAGAGAAGACAAAATCCATTTCCGAAGAGAAGGACTTGAGTGAAAAAGTTGGAAATTTACGTGTTGATTCAGAGGGTGAATCAGCTGATCCTGCCCCTGCAAGTTCCCCCGACGGTAAGAAAGCAACATTGACAGCGGATAGTGAAAGTGACGATGACGACAATAAGAAGAATCCTGAGTATGCTCGGCTTATCACTATCCACCAATCGAAGCCTTCGAAAGGAACTACCGTGGAAGACAGACTGAAAGTTGAAGGGACAGTGGTACGGATTAGTCTTTCAGAGACTAAGCTGGAGAAGGTCACTGAAGAGTTTCCTGAACTTGTGGTCAAGTTCACGCAGAGGAACATTCTACGTGTGTATCCTGCTGGTAACCGAGTAAACTCGTCCAACTATGATCCTACTGCGGCTTGGATTCACGGAGCTCAAATGGTGGCTCAAAATATGCAAGGTTATGGCAAAGAGCTCTGGCAAGCCCACGGCAAGTTCAGGGGAAATGGTGGCTGTGGATACATCCTTAAGCCAAAGTATCTATTGGAAGATTTGCCCAATGGTAAACCTTTTAACCCTTCAGGACAAAGCGTTCCCCAAATACCATTTCGACCTTTTCTCGCCTCCAGATTTCTTCACTAGGCTGCTTGTGACTGGAGTGCCTGCCGATGTGGCAAAGTGGAAAACTTCCGTTATAGATGACGTTTGGGAACCCCACTGGAACGAGGATCACGAGTTTTACCTTAAATGCCCTGAACTTGCACTGCTCCGAATTGAAGTTAGAGATCACGACGAGGAAAGTCAAGATGAGTTCGAAGGGCAGGCGTGCCTTCCAATGCATGAAATTAAAGACGGCTATCGATGCGTGCAGATGTATGACAAAAAGGGCAGTGTGTTGAAGGGCGTGAAAATGTTGTTCCATTTTCAAAAACGTTCGTTTTCTCCGGTCCAGTAATTCATCGTTTTCAAAAACATTTCTGTCTCAATCCTGCAACGAGTAATTTTAGAGAAGGAAGGCTAACGCACTCGAGATGTCTGAAAGGTGAAGAGTTGGAGGGAAGAAGGGTAGCTCGCTGTATCACCGAGCTCTAGTGAACTTCAGGTGTCATTCTTTGAGGGCAGCTCATCTTTCTCATGCATGTGGAACGCTGAGGTGTTGGTTAACGC Nucleotide sequence of the full-length PLC-2from Physcomitrella patens (SEQ ID NO:7)ATCCCGGGCTTCGGGAGTTTAAGAGGATGTCACGGCGTGGGAAGACGAGGCGGTGATGCAGGTTTGGGTGGAGCTTAAGGTTGACGGAGTGTAAGGGATCGGCTCGTCACTGGGTTTGCAAAATGTGTTCCATAGCATGTTGTCGAAGTGGAACCCCGAAAGGGGATCCGGAGCAAGACCTGGTGGGGGAGGTGTTCACAATATACAGCGAGAATGAGAGGATGAGTGCGGAGGGGTTGCTGAAATTCTTGCATACAGAGCAAGGGGATGTCGACTTCACCCTTGATGACGCCAAGCAGATCATGGAGCGCATTCGCAAGGACTGGAAGAAATCCTTCGGACTCGCCTCTATCAACTCAGACTTGTCGAAGGAGGCTTTTCGGAAGTACTTGATGAATCCCGACTTGAATGGCGTCTTACACAACGTTGTTCACCAAGACATGACGCAGCCGATGTCGCACTATTTCATATTCACGGGCCATAACTCGTACCTGACCGGCAACCAGCTGAGCAGCGACAGCAGCGACACACCCATCGCTGCGGCACTGCGGCGCGGCGTGCGGGTTGTGGAATTGGACTTGTGGCCTGATGACAAAGGCGGCATGAAGGTCACACACGGAAACACACTTACCAATCCGGTGTCGTTCCAAAAGTGTGTCACAGCCATCAAGAATAACGCCTTCTTCACCTCGGAGTACCCAGTTTGCGTTACTATTGAGGATCATCTTACAAGCGAATTACAGGGCCATGCTGCAGAGATTTTAGAGCAAATTCTCGGAGACGCCCTGTATTATCCACCCACAACTGATGCATTAGTGGAGTTTCCTTCACCGGAGTCACTGAAGAGGAAGATCATAATCTCCACCAAACCGCCGAAGGAGTATCTCGAAGCATGTTCCACGCAGAAATTGGCCATGGAGAACAGGAATCTGGTGGAGGAGCTTGAGAAGGAAGACAAATTGGAGCAGACCACATTCGCTCCCCTTGAAGAGAACCACATCCTGGGAGAAAATACACCATCGCTGCGTAAGGAAGTCGAGGTTTTAAGCCAAAAGGAAATGTCAACACCAGCTGAGCTTAACTCTAGAAGTCCCTCTGACCTCGGGGAAGCAACATCCACAAGGTATAGCAAGAGCAACGATGGCAATGACAACCCTAAACATTTCAAGTATGCCCGGCTCATCACAATCCGGCTAGCAAAGCACGCAAAGGGGACATCGATGGAGCATCGACTGCAAGTCGATGAATCAGTGAAACGGATCAGTCTGTCGGAATCGAAGCTGGAAAAAGTGGTGGAAAAGTGGCCCGAAGCTCTGGTCAAATTCACGCAGAAGAACATTTTACGTGTGTATCCTGCTGCTAATCGTGTAAACTCCTCCAACTTCTGCCCTACTCTGGCTTGGAACTACGGAGCTCAAATGGTGGCTCAAAACATGCAGGGCTATGGTAAAGAGCTTTGGCAGGCATTTGGCAAGTTCAAGGGAAATGGGGGATGTGGGTATGTTTTGAAGCCACAGTATCTGTTGGAAAACTTGCCTTCTGGTGTGCCTTTCAACCCCACATCACCCAGAAACACAACCCTAATTCTCAAGATTAAAGTTATGACTACCTTGGGATGGGACAAGGCCTTTTCCAAACGCCATTTTGACCTATTCTCACCTCCAGATTTCTTCACTAGGGTGATTGTGGTGGGAGTGCCTGCTGACGAGGCCAAGTGGAAGACATCTGTGGTGGACAATTCATGGGCACCCCATTGGAATGAGGACCATGAGTTTGCCCTAAAATGCCCTGAGCTCGCACTACTTCGCATCGAGGTCCGAGACCATGATGATGATAGCAAAGATGAGTTTGAAGGGCAGACATGCCTTCCCATCCATGAAGTCCGGGATGGGTATCGGTGCATGCAAATGTACGACAAGAAGGGCAATGTACTGAAAGGCGTGCTGATGTTGTTTCATTTTCAAAAGTGCAAATGCACCTTTCAAGACACAGCTCCTATATCCTCTTAAACTCAACCCGCCCACACATGGCCCCATTATCAATTACTAATGCTGCTTTTTATGTTGCCATTGTCATATAATTGTTGGTTTGTGGGGGGGAAGACTGACCAGTTTAGTGTGTGCACCCAAGGTTAACGCC Nucleotide sequence offull-length 14-3-3P-1 from Physcomitrella patens (SEQ ID NO:8)ATCCCGGGCGGACTGTCGTGGACGATGTGCTAGGCCAAGATGAGTACGGAGAAGGAGCGCGAGAGCTATGTGTACATGGCCAAGCTCGCCGAGCAGGCGGAGCGTTACGATGAGATGGTGGAATCGATGAAGAAGGTTGCCAAGCTTGATGTGGAGCTGACAGTAGAGGAGCGAAATCTCTTGTCCGTGGGTTATAAGAATGTCATCGGAGCCCGGAGGGCGTCATGGCGGATCATGTCATCCATCGAACAGAAGGAGGAGAGCAAAGGTAACGAACAGAATGTTAAACGCATCAAGGACTACAGACACAAGGTGGAGGAGGAGCTGTCGAAGATCTGCAATGATATCCTGTCTATCATCGACGGACACCTGATTCCGTCGTCCAGCACGGGAGAGTCCACTGTGTTCTACTATAAAATGAAGGGAGATTACTATCGGTACCTGGCGGAGTTCAAGACCGGGAATGAGAGGAAAGAGGCCGCTGACCAATCTTTGAAGGCATACCAGGCTGCATCCAGCACTGCAGTGACGGACCTGGCACCGACGCATCCTATCCGACTGGGATTAGCTTTGAACTTCTCGGTCTTTTATTATGAAATTTTGAACTCTCCTGAGAGGGCATGCCATTTGGCGAAACAAGCATTTGACGAGGCGATTGCTGAGTTGGATACGTTAAGTGAGGAGTCGTACAAGGACAGCACATTGATCATGCAGCTACTTAGAGATAATCTGACCCTGTGGACATCTGACCTTCAGGACGAGGGAGGTGACGACCAGGGAAAGGGAGATGATATGAGGCCCGAGGAGGCTGAGTGATGACGATTAGGTCTTTTATGTGGAGACGAATTTGCAAATCACTTCACTCAATTGGTGGTGGGCCGGGGCAAGAAGATGTGCAGT TGCGTGCCGAGCTCGCNucleotide sequence of full-length 14-3-3P-2 from Physcomitrella patens(SEQ ID NO:9) GCGTTAACTTCACAATGACGGAGCTACGAGAGGAAAATGTGTACATGGCTAAGCTCGCCGAGCAGGCGGAGCGGTACGATGAGATGGTGGAAGCCATGGAGAATGTGGTAAAGGCGGTGGAGAACGAGGAGCTGACCGTGGAGGAGCGGAACCTGTTGTCGGTGGCGTTTAAGAACGTGATTGGTGCGAGGAGGGCGTCGTGGCGGATCATCTCTTCCATCGAGCAGAAGGAAGAGGCCAAGGGGTCTGAGGAGCACGTCGCTGCTATTAAGGAGTACCGATCCAAAGTAGAGGCTGAGTTGAGCACCATCTGTGACACTATATTGAAGCTTTTGGACTCGCACCTGATCCCGTCCTCCACCTCGGGGGAGTCGAAGGTTTTTTACTTGAAAATGAAGGGAGACTATCACAGGTACCTGGCTGAGTTCAAAGCCGGCGCTGAGAGAAAAGAGGCAGCTGAGGCTACATTGCACGCGTACAAGCATGCACAAGACATTTCAACGACAGAGTTGGCGTCCACACATCCTATCAGATTGGGATTGGCTTTGAATTTTTCTGTCTTTTACTACGAAATTTTGGTTTCTCCAGACCGAGCATGCCATCTTGCCAAGCAGGCTTTTGATGAAGCTATCTCTGAGTTAGATACCTTAGGAGAGGAATCCTACAAAGACAGTACTCTCATTATGCAGCTGCTCCGGGATAACCTTACCTTATGGACTTCAGATATGCAGGACGACATTGGTGAAGAAGGAAAGGATTCCAAGGTCGAAGATGCTGATGACCACTAGAACTTAGAATAGAAACTCATGTCCAGGCGACTAAGTTGAGGCTGGAGCTCGC Nucleotide sequence of thefull-length CBP-1 from Physcomitrella patens (SEQ ID NO:10)ATCCCGGGTCAGCTCGTGGAAGTGTTGCAGCAGCGCGGACGGGCAGGATCGGACATTTTGAGATTTTTGACAGGGCTATCAGAGGTGTTCAGAAGGGACGACAAAGACCAGCATGTCAACAGAGGGAGGACTGCATGTTCTTGATGGATCTCAGATCAGAAATGCATTACCCGATCTTCAATCGAGGAACAGTTTTTCTAAGAATGATGAAGGGTCGAAAGGGTATCTGACACCATCTGAGATGCGGCAGGCTGCGGAAGCAGAAGCAGCCGCTCTTCTCTTAGGTGTCCAACTTTCCTCAAAGATTTTTGAAAATGCTGCATCAAAACTTCCAACTGAAGATTCTGCAGAGATCACGGAGGACGTGTTTTCCAGTACTCTGCAGAGTTATCTAACAGCAATTGCTGATGCTTTAGAAGATGAGCCGGTAGTAGTGTCAGTACTAGACGGCTCAGCAATTAAGGCTTTGCTAGAAGATGAAGATGATTTTGCGATGGTTGCCGAGGATCTGTTTGAGAAGTTAGACACTGATGAGAGTGGCAAGCTGAGCAGCAAAGAGCTTCGACCTGCCATTATGCAGCTGGGCGTTGAGCAGGGTGTCCCTCCTGCCGCAGCTACTACTGAAGCGGAGGAATTGGTTACCAAGTTGATCAACAAGTACGGCCAGGGAACCGAGGAGCTTGGACAAGCTCAATTTGCTGCATTATTGCAAGATGTCCTTCAGGATATGGCCGAGTCTCTTGCAGAGAAACCTATCACAATTGTACGAGATGTGAAAATGCTCAACGGCTCTCATTTGCGAAAGATGCTGGCTGATGAGAAGGCATTCAAGGAAATGGCAGATAACATGTTTAATGACCTAGATGTCAACAAAGATCAACGCTTGAGCAAGGCTGAAATCAGACCATTATTTGAACAACAAACTGCAGCGTGGGGTCTACCTCCCGTTGGTGATTCGGACACAGAAGAACTATTTGACGAGGTTTTCAAGGCCGTTGACTCAGACAAAAGTGGGGAAGTTGAAAAGCCTGAGTTTGCAGTTCTTGTCAAGACTCTCCTTGCGGATTTTGCGGAAACGTTGCGGCTCAACCCAATACTAGTGGAGATAGAAACTGCCTCTCGTTGAAGCATCGAGTCATAGTTCTGGGGGAGCGATGTTTTCTAAAGTCGTAGTCCATTTTTGGATAAGATGACTTTGCACCCAGAGTTCTTGTGAAACGTGACACCAGGTATGATGAAGGCTTGATGATATTTTAGAGTGACAATTTTATGTGGCTAGAGGCTCATGAGGTCGTGAGATCGAAGTGGAAATGATTTGTGAAAGCTACTTCGACCTGGGTAGCTTTTCTAAGCTAGGATAGTTATATGAAAAAGATAATTAAACTTCAAGCGGATCAATATAGCTCACAGAATCCATTCTTCGTTTCTGTTTCCTGAGAACCCACCAATGTCCAAGTTACAAAACTCCGTGGGAGAAACAGACGTGCAGTGCATGCATAAGGTTGGTGTGATTGTTTGCGTAGTGATGTTTCTGGATGACTTGAATAGAATCAAGTGCATAGATAGTCAATTGTCTCACACAAGATCTTCGAACAATCCACCAACCGGCGTTGCCAGTCGTGCGGAGGGCACGGTTGGTGGGACGGACTAGCGGTGCGACGCGTGTAGAGAATGCATTGGCGGCTGCAGATTAGACAGTTGTTCGCATCCGTTGTAGGATAGATCGTTAGGATACTCGACTCTTACCTGTGTTCGAATTCCGGCATCGGAAGCCCCCGAGTGAAAATTGGACGAGCTCGC Deduced amino acidsequence of PLC-1 from Physcomitrella patens (SEQ ID NO:11)MCSIPFGRKKSKKGDLAQDLLGDVFSTYSENGKLDAEGLLKFLQTEQGDSKSSLDDAKHLVELIRNERHKSKFPGFIVSSDLSKGDFKNYVLSPDLNGVLESTVHQDMTQPLSHYFIFTGHNSYLTGNQLSSDSSDVPIAAALQRGVRVVELDLWPDDKGGIKVTHGNTLTSPVAFEKCIKAIKANAFVSSKYPVVITLEDHLSSPLQALAAETLTNILGEDLYYPPSSDGFKELPSPESLKGKILISTKPPKEYLEAAVAQKSALKDEKILNEFKKADKLQEQSTAPVKSPVEKKIAVPPSEKTKSISEEKDLSEKVGNLRVDSEGESADPAPASSPDGKKATLTADSESDDDDNKKNPEYARLITIHQSKPSKGTTVEDRLKVEGTVVRISLSETKLEKVTEEFPELVVKFTQRNILRMCSIPFGRKKSKKGDLAQDLLGDVFSTYSENGKLDAEGLLKFLQTEQGDSKSSLDDAKHLVELIRNERHKSKFPGFIVSSDLSKGDFKNYVLSPDLNGVLESTVHQDMTQPLSHYFIFTGHNSYLTGNQLSSDSSDVPIAAALQRGVRVVELDLWPDDKGGIKVTHGNTLTSPVAFEKCIKAIKANAFVSSKYPVVITLEDHLSSPLQALAAETLTNILGEDLYYPPSSDGFKELPSPESLKGKILISTKPPKEYLEAAVAQKSALKDEKILNEFKKADKLQEQSTAPVKSPVEKKIAVPPSEKTKSISEEKDLSEKVGNLRVDSEGESADPAPASSPDGKKATLTADSESDDDDNKKNPEYARLITIHQSKPSKGTTVEDRLKVEGTVVRISLSETKLEKVTEEFPELVVKFTQRNILRVYPAGNRVNSSNYDPTAAWIHGAQMVAQNMQGYGKELWQAHGKFRGNGGCGYILKPKYLLEDLPNGKPFNPSAPGDTKMILKVKVMTTMGWDKAFPKYHFDLFSPPDFFTRLLVTGVPADVAKWKTSVIDDVWEPHWNEDHEFYLKCPELALLRIEVRDHDEESQDEFEGQACLPMHEIKDGYRCVQMYDKKGSVLKGVKMLFHFQKRSF SPVQ Deduced aminoacid sequence of PLC-2 from Physcomitrella patens (SEQ ID NO:12)MCSIACCRSGTPKGDPEQDLVGEVFTIYSENERMSAEGLLKFLHTEQGDVDFTLDDAKQIMERIRKDWKKSFGLASINSDLSKEAFRKYLMNPDLNGVLHNVVHQDMTQPMSHYFIFTGHNSYLTGNQLSSDSSDTPIAAALRRGVRVVELDLWPDDKGGMKVTHGNTLTNPVSFQKCVTAIKNNAFFTSEYPVCVTIEDHLTSELQGHAAEILEQILGDALYYPPTTDALVEFPSPESLKRKIIISTKPPKEYLEACSTQKLAMENRNLVEELEKEDKLEQTTFAPLEENHILGENTPSLRKEVEVLSQKEMSTPAELNSRSPSDLGEATSTRYSKSNDGNDNPKHFKYARLITIRLAKHAKGTSMEHRLQVDESVKRISLSESKLEKVVEKWPEALVKFTQKNILRVYPAANRVNSSNFCPTLAWNYGAQMVAQNMQGYGKELWQAFGKFKGNGGCGYVLKPQYLLENLPSGVPFNPTSPRNTTLILKIKVMTTLGWDKAFSKRHFDLFSPPDFFTRVIVVGVPADEAKWKTSVVDNSWAPHWNEDHEFALKCPELALLRIEVRDHDDDSKDEFEGQTCLPIHEVRDGYRCMQMYDKKGNVLKGVLMLFHFQKCKCTFQDTAPISS Deduced amino acid sequence of 14-3-3P-1from Physcomitrella patens (SEQ ID NO:13)MSTEKERESYVYMAKLAEQAERYDEMVESMKKVAKLDVELTVEERNLLSVGYKNVIGARRASWRIMSSIEQKEESKGNEQNVKRIKDYRHKVEEELSKICNDILSIIDGHLIPSSSTGESTVFYYKMKGDYYRYLAEFKTGNERKEAADQSLKAYQAASSTAVTDLAPTHPIRLGLALNFSVFYYEILNSPERACHLAKQAFDEAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDLQDEGGDDQGKGD DMRPEEAE Deducedamino acid sequence of 14-3-3P-2 from Physcomitrella patens (SEQ IDNO:14) MTELREENVYMAKLAEQAERYDEMVEAMENVVKAVENEELTVEERNLLSVAFKNVIGARRASWRIISSIEQKEEAKGSEEHVAAIKEYRSKVEAELSTICDTILKLLDSHLIPSSTSGESKVFYLKMKGDYHRYLAEFKAGAERKEAAEATLHAYKHAQDISTTELASTHPIRLGLALNFSVFYYEILVSPDRACHLAKQAFDEAISELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQDDIGEEGKDSK VEDADDH Deduced aminoacid sequence of CBP-1 from Physcomitrella patens (SEQ ID NO:15)MSTEGGLHVLDGSQIRNALPDLQSRNSFSKNDEGSKGYLTPSEMRQAAEAEAAALLLGVQLSSKIFENAASKLPTEDSAEITEDVFSSTLQSYLTAIADALEDEPVVVSVLDGSAIKALLEDEDDFAMVAEDLFEKLDTDESGKLSSKELRPAIMQLGVEQGVPPAAATTEAEELVTKLINKYGQGTEELGQAQFAALLQDVLQDMAESLAEKPITIVRDVKMLNGSHLRKMLADEKAFKEMADNMFNDLDVNKDQRLSKAEIRPLFEQQTAAWGLPPVGDSDTEELFDEVFKAVDSDKSGEVEKPEFAVLVKTLLADFAETLRLNPILVEIETASR

1. A transgenic plant cell transformed with an expression cassettecomprising an isolated polynucleotide selected from the group consistingof: a) a polynucleotide having a sequence comprising nucleotides 1 to1800 of SEQ ID NO:10; and b) a polynucleotide encoding a polypeptidehaving a sequence comprising amino acids 1 to 337 of SEQ ID NO:15. 2.The plant cell of claim 1, wherein the polynucleotide has the sequencecomprising nucleotides 1 to 1800 of SEQ ID NO:10.
 3. The plant cell ofclaim 1, wherein the polynucleotide encodes the polypeptide having thesequence comprising amino acids 1 to 337 of SEQ ID NO:15.
 4. Atransgenic plant transformed with an expression cassette comprising anisolated polynucleotide selected from the group consisting of: a) apolynucleotide having a sequence comprising nucleotides 1 to 1800 of SEQID NO:10; and b) a polynucleotide encoding a polypeptide having asequence comprising amino acids 1 to 337 of SEQ ID NO:15.
 5. The plantof claim 4, wherein the polynucleotide has the sequence comprisingnucleotides 1 to 1800 of SEQ ID NO:10.
 6. The plant of claim 4, whereinthe polynucleotide encodes the polypeptide having the sequencecomprising amino acids 1 to 337 of SEQ ID NO:15.
 7. The plant of claim4, wherein the plant is a monocot.
 8. The plant of claim 4, wherein theplant is a dicot.
 9. The plant of claim 4, wherein the plant is selectedfrom the group consisting of maize, wheat, rye, oat, triticale, rice,barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper,sunflower, tagetes, potato, tobacco, eggplant, tomato, Vicia species,pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut,perennial grasses, and a forage crop plant.
 10. The plant of claim 9,wherein the plant is maize.
 11. The plant of claim 9, wherein the plantis soybean.
 12. The plant of claim 9, wherein the plant is rapeseed orcanola.
 13. The plant of claim 9, wherein the plant is cotton.
 14. Aseed which is true breeding for a transgene comprising a polynucleotideselected from the group consisting of: a) a polynucleotide having asequence comprising nucleotides 1 to 1800 of SEQ ID NO:10; and b) apolynucleotide encoding a polypeptide having a sequence comprising aminoacids 1 to 337 of SEQ ID NO:15.
 15. The seed of claim 14, wherein thepolynucleotide has the sequence comprising nucleotides 1 to 1800 of SEQID NO:10.
 16. The seed of claim 14, wherein the polynucleotide has thesequence encoding the polypeptide having the sequence comprising aminoacids 1 to 337 of SEQ ID NO:15.
 17. An isolated polynucleotide selectedfrom the group consisting of: a) a polynucleotide having a sequencecomprising nucleotides 1 to 1800 of SEQ ID NO:10; and b) apolynucleotide encoding a polypeptide having a sequence comprising aminoacids 1 to 337 of SEQ ID NO:15.
 18. The isolated nucleic acid of claim17, wherein the polynucleotide has the sequence comprising nucleotides 1to 1800 of SEQ ID NO:10.
 19. The isolated nucleic acid of claim 17,wherein the polynucleotide encodes the polypeptide having the sequencecomprising amino acids 1 to 337 of SEQ ID NO:15.
 20. A method ofproducing a drought-tolerant transgenic plant, the method comprising thesteps of: a) transforming a plant cell with an expression cassettecomprising a polynucleotide selected from the group consisting of: i) apolynucleotide having a sequence comprising nucleotides 1 to 1800 of SEQID NO:10; and ii) a polynucleotide encoding a polypeptide having asequence comprising amino acids 1 to 337 of SEQ ID NO:15; b) growing thetransformed plant cell to generate transgenic plants; and c) screeningthe transgenic plants generated in step b) to identify a transgenicplant that expresses the polypeptide and exhibits increased tolerance todrought stress as compared to a wild type variety of the plant.
 21. Themethod of claim 20, wherein the polynucleotide has the sequencecomprising nucleotides 1 to 1800 of SEQ ID NO:10.
 22. The method ofclaim 20, wherein the polynucleotide has the sequence encoding thepolypeptide having the sequence comprising amino acids 1 to 337 of SEQID NO:15.